JP6818849B2 - Nuclear transport regulators and their use - Google Patents

Description

The present invention relates to nuclear transport regulators and their use.

Mutual Reference of Related Applications This application is a US Provisional Patent Application No. 61 / 644,802 filed on May 9, 2012, and a US Provisional Patent Application No. 61 filed on March 15, 2013. / 798, 188 claims priority. The entire teachings of the above application are incorporated herein by reference.

Cells from most major human solid and hematological malignancies exhibit aberrant cell localization of a variety of carcinogenic proteins, tumor suppressor proteins, and cell cycle regulators (Cronshaw et al. 2004, Falini et al. 2006). ). For example, certain p53 mutations result in localization into the cytoplasm rather than in the nucleus. This results in the loss of normal growth control, even though the tumor suppressor function is intact. In other tumors, wild-type p53 is sequestered in the cytoplasm or rapidly degraded, again leading to loss of its suppressor function. Restoration of proper nuclear localization of the functional p53 protein can normalize some properties of neoplastic cells (Cai et al. 2008; Hoshino et al. 2008; Rain et al. 1999a; Rain et al. 1999b; Smart et al. 1999), which could restore the susceptibility of cancer cells to DNA damaging agents (Cai et al. 2008) and regress established tumors (Sharpless & DePinho 2007, Xue et al. 2007). Can bring. Similar data are available for other tumor suppressor proteins such as Forkhead (Turnerand Sullivan 2008) and c-Abl (Vignari and Wang 2001). In addition, aberrant localization of some tumor suppressors and growth control proteins may be involved in the pathogenesis of autoimmune diseases (Davis 2007, Nakahara 2009). Inhibition of CRM1 is caused by familial cancer syndrome (eg, Lee Fraumeni syndrome, BRCA1 or BRCA2) due to a deficiency of one p53 allogeneic gene, in which the specific tumor suppressor protein (TSP) is deleted or dysfunctional. Cancer syndrome) may provide a particularly interesting benefit, and increased TSP levels by systemic (or topical) administration of CRM1 inhibitors can help restore normal tumor suppressor function.

Certain proteins and RNA are carried into the nucleus by specialized transport molecules, which are classified as importins when they carry the molecule into the nucleus and are classified as exportin when they carry the molecule out of the nucleus. , Then carried out (Terry et al. 2007; Sorokin et al. 2007). Proteins that are carried into and out of the nucleus contain a nuclear carry-in / localization (NLS) or carry-out (NES) sequence that allows them to interact with a valid transporter. Chromosome Region Maintenance 1 (Crm1), also referred to as export-1 or Xpo1, is the major exportin.

Overexpression of Crm1 includes human ovarian cancer (Nosuke et al. 2008), cervical cancer (van der Watt et al. 2009), pancreatic cancer (Hung et al. 2009), and hepatocellular carcinoma (Pascale et). It has been reported in several tumors, including al. 2005) and osteosarcoma (Yao et al. 2009), and has been independently associated with poor clinical outcomes of these tumor types.

Inhibition of Crm1 is associated with gene expression, cell proliferation, angiogenesis, and epigenetics, p53, c-Abl, p21, p27, pRB, BRCA1, IkB, ICp27, E2F4, KLF5, YAP1, ZAP, Blocks the mass outflow of tumor suppressor proteins and / or growth regulators such as KLF5, HDAC4, HDAC5 or forkhead proteins (eg FOXO3a) from the nucleus. Crm1 inhibitors have been shown to induce apoptosis in cancer cells, even in the presence of carcinogenic activation or growth-promoting signals, while leaving normal (non-transformed) cells. The majority of studies of Crm1 inhibition utilize the natural Crm1 inhibitor leptomycin B (LMB). LMB itself is highly toxic to neoplastic cells, but is poorly tolerated and has significant gastrointestinal toxicity in animals (Roberts et al. 1986) and humans (Newlands et al. 1996). Derivatization of LMB to improve drug-like properties results in better tolerated compounds that maintain antitumor activity in animal tumor models (Yang et al. 2007, Yang et al. 2008, Mutka et al. .2009). Thus, nuclear export inhibitors can have beneficial effects in neoplasms and other proliferative disorders. However, until now, small molecule drug-like Crm1 inhibitors for in vitro and in vivo use have been rare.

In addition to the tumor suppressor protein, Crm1 also carries out some important proteins involved in the inflammatory process. These include IkB, NF-kB, Cox-2, RXRα, Commd1, HIF1, HMGB1, FOXO, FOXP, and others. The nuclear factor κB (NF-kB / rel) family of transcriptional activators, named after the discovery that it drives immunoglobulin κ gene expression, is diverse, involved in inflammation, proliferation, immunity, and cell survival. Modulates mRNA expression of various genes. Under basic conditions, a protein inhibitor of NF-kB called IkB binds to NF-kB in the nucleus and the complex IkB-NF-kB inactivates NF-kB transcriptional function. In response to inflammatory stimuli, IkB dissociates from the IkB-NF-kB complex, which releases NF-kB and exposes its potent transcriptional activity. Numerous signals that activate NF-kB activate NF-kB by targeting IkB for proteolysis (phosphorylation of IkB ubiquitinates it and then "labels" it for proteolysis. Do "). The nuclear IkBa-NF-kB complex can be carried into the cytoplasm by Crm1 where it dissociates and NF-kB can be reactivated. Ubiquitinated IkB may also dissociate from the NF-kB complex to restore NF-kB transcriptional activity. Inhibition by LMB of Crm1-induced delivery to human neutrophils and macrophage-like cells (U937) not only results in the accumulation of transcriptionally inactive nuclear IkBa-NF-kB complexes, but also during cell stimulation. It also interferes with the initial activation of NF-kB (Gosh 2008, Huang 2000). In another study, treatment with LMB was performed in lung microvascular endothelial cells with IL-1β-induced NF-kBDNA binding (the first step in NF-kB transcriptional activation), IL-8 expression, and intercellular adhesion molecules. Expression was inhibited (Walsh 2008). COMMD1 is another nuclear inhibitor for both NF-kB and hypoxia-inducing factor 1 (HIF1) transcriptional activity. Blocking nuclear exports of COMMD1 by inhibiting Crm1 results in increased inhibition of NF-kB and HIF1 transcriptional activity (Muller 2009).

Crm1 also mediates retinoid X receptor α (RXRα) transport. RXRα is highly expressed in the liver and plays a central role in the regulation of bile acids, cholesterol, fatty acids, steroids, and foreign body metabolism, and homeostasis. During liver inflammation, nuclear RXRα levels are significantly reduced, primarily due to the inflammation-mediated nuclear exports of RXRα by Crm1. Lep B can prevent an increase in RXRα levels in the IL-1β-induced cytoplasm in human liver-derived cells (Zimmerman 2006).

The role of Crm1-mediated extranuclear export in NF-kB, HIF-1, and RXRα signaling is to block extranuclear export of the vasculature (vasculitis, arthritis, rheumatic polymyositis, atherosclerotic). Arteriosclerosis), dermatological (see above), rheumatic (rheumatic and related arthritis, psoriatic arthritis, spondyloarthropathies, crystalline arthropathy, systemic erythematosus, mixed connective tissue disease, myitis syndrome, dermatomyositis) , Encapsulation myositis, undifferentiated connective tissue disease, Sjogren's syndrome, rheumatism, and duplication syndrome), suggesting that it can be potentially beneficial in numerous inflammatory processes across multiple tissues and organs. To do.

Inhibition of CRM1 affects gene expression by inhibiting / activating a series of transcription factors such as ICp27, E2F4, KLF5, YAP1, ZAP.

Inhibition of Crm1 for a number of dermatological syndromes, including inflammatory skin diseases (atopy, allergic dermatitis, chemical dermatitis, psoriasis), sunburn damage (ultraviolet / UV damage), and infections. It has a potential therapeutic effect. Inhibition of CRM1, best studied with LMB, showed minimal effect on normal keratinocytes and exerted anti-inflammatory activity against keratinocytes exposed to UV, TNFa, or other inflammatory stimuli (Kobayashi & Shinkai 2005). , Kannan & Jaiswal 2006). Inhibition of Crm1 also protects NRF2 (nuclear factor erythrocyte system-related factor 2) activity from oxidative damage to keratinocytes (Schafer et al. 2010, Kannan & Jaiswar 2006) and other cell types (Wang et al. 2009). Control upward. LMB induces apoptosis in keratinocytes infected with carcinogenic human papillomavirus (HPV) strains such as HPV16, but not in uninfected keratinocytes (Jolly et al. 2009).

Crm1 also mediates the transport of important neuroprotective proteins that may be useful in neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease, and amyotrophic lateral sclerosis. For example, (1) NRF2 (Wang 2009), FOXA2 (Kittappa et al. 2007) and other important neuroprotective regulators are forced to retain in the nucleus and / or (2) glial cells. By inhibiting NFκB transcriptional activity by isolating IκB into the nucleus, inhibition of Crm1 can delay or prevent the neuronal cell death seen in these disorders. There is also evidence to link abnormal glial cell proliferation to abnormal CRM1 levels or CR1 function (Shen 2008).

An intact nuclear export, primarily mediated through CRM1, is also required for the intact maturation of many viruses. Viruses that are associated with nuclear export and / or CRM1 itself in their life cycle include human immunodeficiency virus (HIV), adenovirus, monkey retrovirus type 1, Borna disease virus, and influenza ( Normal strain and H1N1 and avian H5N1 strain), B (HBV) and C (HCV) hepatitis virus, human papillomavirus (HPV), respiratory symptom virus (RSV), Dungee, severe acute respiratory syndrome coronavirus , Yellow fever virus, West Nile virus, simple herpesvirus (HSV), cytomegalovirus (CMV), and Mercel cell polyomavirus (MCV). (Bhuvanakanzam 2010, Cohen 2010, Whitetaker 1998). Additional viral infections that rely on undamaged nuclear exports are likely to be discovered in the near future.

The HIV-1 Rev protein, which travels through the nucleolus and back and forth between the nucleus and cytoplasm, is an HIV transcript that contains Rev Response Element (RRE) RNA, is not spliced, and is individually spliced. Facilitates unloading through the CRM1 unloading route. Inhibition of Rev-mediated RNA transport using CRM1 inhibitors such as LepB or PKF050-638 arrests the HIV-1 transcription process and inhibits the production of new HIV-1 virions, thereby lowering HIV-1 levels. Obtain (Pollard 1998, Daelemans 2002).

Dengue virus (DENV) is the pathogen of dengue fever (DF), a common arthropod-mediated viral disease, and its more severe and potentially lethal dengue hemorrhagic fever (DHF). DHF appears to be the result of an excessive inflammatory response to DENV. NS5 is the largest and best conserved protein of DENV. CRM1 regulates NS5 transport from the nucleus to the cytoplasm where most of the functions of NS5 are mediated. CRM1-mediated inhibition of NS5 export results in altered viral production kinetics, reduces induction of inflammatory chemokine interleukin-8 (IL-8), DENV, and other, including hepatitis C virus. We present new means for the treatment of diseases caused by the medically important flavivirus (Rawlinson 2009).

Other virus-encoded RNA-binding proteins that use CRM1 to exit the nucleus include HSV1-type tegment protein (VP13 / 14, or hUL47), human CMV protein pp65, SARS coronavirus ORF 3b protein, And RSV matrix (M) proteins include (Williams 2008, Sanchez 2007, Freundt 2009, Gildyal 2009).

Interestingly, many of these viruses include hepatocellular carcinoma (HCC) due to chronic HBV or HCV infection, cervical cancer due to HPV, and Merkel cell carcinoma associated with MCV. Is associated with a particular type of human cancer. Thus, CRM1 inhibitors can have beneficial effects on both the viral transmission process and the malignant transformation process resulting from these viruses.

CRM1 regulates nuclear localization and thus the activity of multiple DNAs that metabolize enzymes such as histone deacetylase (HDAC), histone acetyltransferase (HAT), and histone methyltransferase (HMT). .. Suppression of cardiomyocyte hypertrophy by irreversible CRM1 inhibitors has been demonstrated and is associated with nuclear retention (and activation) of HDAC5, an enzyme known to suppress hypertrophic genetic programs. It is possible (Monovich et al. 2009). Thus, CRM1 inhibition may have beneficial effects in hypertrophic syndromes, including certain forms of congestive heart failure and hypertrophic cardiomyopathy.

CRM1 has also been associated with other disorders. Reaver's disease, a hereditary disease characterized by degeneration of retinal ganglion cells and loss of vision, has been associated with CRM1 switch inactivity (Gupta N 2008). There is also evidence linking neurodegenerative disorders to abnormal nuclear transport.

Mutka S, Yang W, Dong S, et al. 2009. Identity of nuclear export in vivo. With potency titancer activity in vivo. Cancer Res. 69: 510-7.

In view of the above, it is desirable to discover a compound that regulates nuclear transport.

The present invention uses a compound useful as a nuclear transport regulator, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable composition containing the compound of the present invention, and the composition for treating various disorders. Regarding the method used in. Now, while the nuclear transport regulators of the invention and their pharmaceutically acceptable salts and / or compositions provide the desired in vivo exposure as determined by AUC in mice, compared to other regulators. It was found to show lower levels of brain permeability. The compounds of the present invention have the general formula I:
Or have a pharmaceutically acceptable salt thereof, and in the formula, each variable is as defined and described herein.

The compounds of the invention and pharmaceutically acceptable compositions thereof are useful in the treatment of a variety of diseases, disorders or conditions associated with abnormal cellular responses caused by improper nuclear transport. Such diseases, disorders, or medical conditions include those described herein.

The compounds provided by the present invention also study nuclear transport regulation in biological and pathological phenomena; study of cell communication pathways mediated by such kinases; and comparative evaluation of new nuclear transport regulators. It is also useful for.

The above will become apparent from the following more specific description of the exemplary embodiments of the invention.

That is, the gist of the present invention is
[1] Equation I:
Compound (in the formula,
R ¹ is selected from hydrogen and C _{1 to} C ₄ alkyl;
R ² is selected from O and S;
R ^{3 _{is, -N (R 4) - (}} C 3 ~C 6 cycloalkyl), _- C 1 -C ₆ alkyl, - _(C 0 -C ₄ alkylene) - heterocyclyl, and - _(C 0 -C ₄ alkylene ) - it is selected from heteroaryl (wherein all alkyl, alkylene, a heterocyclyl, and heteroaryl portion of R ³ is independently optionally substituted);
R ⁴ is selected from hydrogen and _C 1 -C ₄ alkyl),
Or its pharmaceutically acceptable salt,
[2] The compound according to [1], wherein R ¹ is selected from hydrogen and methyl.
[3] The compound according to [2], wherein R ¹ is hydrogen.
[4] The compound according to any one of [1] to [3], wherein R ² is O.
[5] wherein R ⁴ is hydrogen, [1] to a compound according to any one of [4],
[6] R ³ is -N (R ⁴ )-(C _{3 to} C ₆ cycloalkyl), -C _{3 to} C ₆ alkyl,-(C _{0 to} C ₁ alkylene) -heterocyclyl, and-(C ₀ to). C ₁ alkylene) - is selected from heteroaryl:
Any alkyl or alkylene moiety of R ³ is optionally and independently substituted with one or more substituents selected from the group consisting of oxo and -N (R ⁵ ) ₂ . Each R ⁵ is selected independently of hydrogen and C _{1 to} C ₄ alkyl;
Any heterocyclyl moiety R ³ is, comprises at least one nitrogen atom in the ring, optionally substituted with one or more substituents selected from the group consisting of C ₁ -C ₄ alkyl and oxo Be;
Every heteroaryl moiety of R ³ comprises at least one nitrogen atom in the ring and is optionally substituted with one or more C _{1 to} C ₄ alkyl.
The compound according to any one of [1] to [5].
[7] The compound according to [6], wherein R ³ is − (C _{0 to} C ₁ alkylene) -heterocyclyl.
[8] The compound according to [7], wherein R ³ is-(C ₁ alkylene) -heterocyclyl.
[9] The compound according to [7] or [8], wherein the heterocyclyl is selected from pyrazineyl, piperidinyl, morpholinyl, and pyrazolyl.
[10] The heterocyclyl is -C (CH ₃ ) ₃ , -NH-cyclopropyl, -CH ₂ -pyrazine-2-yl, -pyrazine-2-yl, -CH ₂ -morpholin-4-yl, and 5 - morpholinyl ^{R 3} is selected from methyl -1-H- pyrazol-4-yl, the compound according to [9],
[11] any alkyl ^{R 3,} alkylene, a heterocyclyl, and heteroaryl moieties are optionally, independently, -OH, -SH, nitro, halogen, amino, _cyano, C 1 _{-C 12} alkyl, C Selected from the group consisting of _{2 to} C ₁₂ alkenyl or C _{2 to} C ₁₂ alkynyl groups, C _{1 to} C ₁₂ alkoxy, C _{1 to} C ₁₂ haloalkyl, C _{1 to} C ₁₂ haloalkoxy, and C _{1 to} C ₁₂ alkyl sulfanyl. Substituted with one or more substituents,
The compound according to any one of [1] to [5].
[12] All alkyl, alkylene, heterocyclyl, and heteroaryl moieties of R ³ are optionally independently and independently of the formula −N (R ⁵ ) ₂ (in which each R ⁵ is hydrogen and C ₁ to C ₁ to independently from C ₄ alkyl is substituted with an amino group having to) selected,
The compound according to any one of [1] to [5].
[13] All the heteroaryl portion of ^{R 3} is optionally, independently, -OH, -SH, nitro, halogen, amino, _cyano, C 1 _{-C 12 alkyl,} C 2 _{-C 12} alkenyl, C One or more substitutions selected from the group consisting of _{2 to} C ₁₂ alkoxy, C _{1 to} C ₁₂ alkoxy, C _{1 to} C ₁₂ haloalkyl, C _{1 to} C ₁₂ haloalkoxy, and C _{1 to} C ₁₂ alkyl sulfanyl. Substituted by group;
Any alkyl, alkylene or heterocyclyl moiety of R ³ is optionally independent, oxo, -OH, -SH, nitro, halogen, amino, cyano, C _1- C ₁₂ alkyl, C _2- C ₁₂ alkenyl. , _C 2 _{-C 12 alkynyl,} C 1 _{-C 12 alkoxy,} C 1 _{-C 12 haloalkyl,} C 1 _{-C 12} haloalkoxy, and _C 1 is selected from _{-C 12} alkylsulfanyl group consisting of Le, one or more Substituted with a substituent of
The compound according to any one of [1] to [5].
[14] R ³ is -N (R ⁴ )-(C _{3 to} C ₆ cycloalkyl), -C _{3 to} C ₆ alkyl,-(C _{0 to} C ₁ alkylene) -heterocyclyl, and-(C ₀ to C ₁ alkylene) -selected from heteroaryl;
Any alkyl or alkylene moiety of R ³ is (wherein each ^{R 5} is, independently from hydrogen and _C 1 -C ₄ alkyl is selected) optionally -N ^(R _{5) 2} is replaced by;
Any heterocyclyl, and heteroaryl portion of R ³ is, comprises at least one nitrogen atom in the ring;
Any heterocyclyl, and heteroaryl portion of R ³ is substituted by optionally _C 1 -C ₄ alkyl,
The compound according to any one of [1] to [5].
[15] R ³ is -C (CH ₃ ) ₃ , -CH (NH ₂ ) -CH (CH ₃ ) ₂ , -NH-cyclopropyl,-(CH ₂ ) _0-1 -pyrazinyl, piperidinyl, hydroxypi Perijiniru, N- methylpiperidinyl, -CH ₂ - morpholin-4-yl, and are selected from methyl pyrazolyl compound according to [14],
[16] R ³ is -C (CH ₃ ) ₃ , -CH (NH ₂ ) -CH (CH ₃ ) ₂ , -NH-cyclopropyl,-(CH ₂ ) _0-1 -pyrazine-2-yl, piperidin-3-yl, -CH ₂ - morpholin-4-yl, and is selected from 5-methyl -1-H- pyrazol-4-yl, the compound according to [15],
[17] R ³ is -C (CH ₃ ) ₃ , -NH-cyclopropyl, -CH ₂ -pyrazine-2-yl, -pyrazine-2-yl, -CH ₂ -morpholin-4-yl, and 5 The compound according to [16], selected from −methyl-1-H-pyrazole-4-yl.
[18] Structural formula,
A compound represented by any one of, or a pharmaceutically acceptable salt thereof,
[19] The compound according to [18], which is selected from any one of compounds 1, 2, 3, 4, 8, 11, 12, and 13.
[20] A pharmaceutical composition comprising the compound according to any one of [1] to [19], or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
[21] A method for treating a CRM1 activity-related disorder, which comprises the step of administering a therapeutically effective amount of the pharmaceutical composition according to [20] to a subject in need thereof.
[22] The disorder is selected from proliferative diseases, inflammatory diseases, autoimmune diseases, viral infections, ophthalmic diseases, neurodegenerative diseases, abnormal tissue growth disorders, food intake-related disorders, allergies, and respiratory diseases. The method according to [21],
[23] The method according to [22], wherein the disease is cancer.
[24] The method according to [23], wherein the cancer is lymphoma.
[25] The method according to [23] or [24], wherein the composition is administered together with a second therapeutic agent useful for treating cancer.
[26] The method according to [21], wherein the disease is arthritis.
[27] The method according to [21], wherein the disease is psoriasis.
[28] The method according to [21], wherein the disease is obesity.
[29] A method of promoting wound healing in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to [20].
[30] The method of [29], wherein the wound is a trauma, a surgical wound, an internal wound, a chronic wound, an ulcer, a burn, or is the result of radiation exposure.
[31] The wound consists of burns, cuts, open wounds, surgical or post-surgical wounds, diabetic lesions, burns, chemical burns, radiation burns, decubitus, rubbing on the floor, and medical conditions associated with diabetes or poor circulation. The method according to [29], which is selected from the group.

The present invention may provide compounds that regulate nuclear transport.

Average tumor volume over time, showing group average volume of Z-138 xenograft tumors in mice treated with vehicle, 80 mg / kg cyclophosphamide, 15 mg / kg Compound 2 or 7.5 mg / kg Compound 2. (The error bar represents the SEM of each group). A graph of mean tumor volume over time showing the group average volume of A549 xenograft tumors in mice treated with vehicle, 5 mg / kg cisplatin, 10 mg / kg Compound 2 or 5 mg / kg Compound 2 (error bar). Represents the SEM of each group). Over a 12-day observation period, showing clinical arthritis scores of anti-collagen antibody-induced male BALB / c arthritis mice treated with vehicle, dexamethasone, 4 mg / kg Compound 2 or 7.5 mg / kg Compound 2, over time. It is a graph of the overall arthritis score (¥ = dexamethasone treatment group that is significantly different from the vehicle treatment group; # = 7.5 mg / kg compound 2 treatment group that is significantly different from the vehicle treatment group; † = vehicle treatment group and Compound 2 treatment group of 4 mg / kg with significant difference). Shows the group mean hindlimb thickness of anti-collagen antibody-induced male BALB / c arthritis mice treated with vehicle, dexamethasone, 4 mg / kg of Compound 2 or 7.5 mg / kg of Compound 2 over a 12-day observation period. , Average hindlimb graph over time (¥ = dexamethasone treatment group with significant difference from vehicle treatment group; # = 7.5 mg / kg compound 2 treatment group with significant difference from vehicle treatment group; † = vehicle treatment group Compound 2 treatment group of 4 mg / kg with significant difference). FIG. 5 is a graph of joint swelling over time showing joint swelling measured on a scale of 0-4 in unsensitized rats and rats treated according to the CIA model with positive controls or Compound 2. FIG. 5 is a graph of clinical scores as a function of time, showing unsensitized rats and rat arthritis scores treated according to the CIA model with positive controls or Compound 2. It is a representative image from each treatment group in the CIA model showing the histopathology of unsensitized rats and rat hind paws, treated according to the model with positive controls or Compound 2. FIG. 5 is a graph of auricular thickness over time showing the group average of left auricular thickness in female BALB / c mice treated with vehicle, PMA and vehicle, PMA and Compound 2, or PMA and betamethasone. FIG. 5 is a graph of auricular thickness over time showing the group average of right auricular thickness in female BALB / c mice treated with vehicle, PMA and vehicle, PMA and Compound 2, or PMA and betamethasone. FIG. 6 is a disease activity graph showing the group average of left auricular disease activity in female BALB / c mice treated with vehicle, PMA and vehicle, PMA and Compound 2, or PMA and betamethasone. FIG. 6 is a disease activity graph showing the group average of disease activity in female BALB / c mice treated with vehicle, PMA and vehicle, PMA and Compound 2, or PMA and betamethasone. Disease activity over time showing disease activity in male BALB / c mice treated with vehicle, IMQ and vehicle, IMQ and 1 μM Compound 2, or IMQ and 10 mg / kg cyclophosphamide prior to IMQ administration. It is a graph of the index. Disease activity index over time, indicating disease activity in male BALB / c mice treated with vehicle, IMQ and vehicle, IMQ and 1 μM Compound 2, or IMQ and 10 mg / kg cyclophosphamide after IMQ administration. It is a graph of. Cumulative food intake over time, indicating cumulative food intake of lean and obese Zucker rats treated with vehicle (VEH), 1.5 mg / kg of compound or 3.0 mg / kg of compound 2. It is a graph. Average food intake over time, indicating the average food intake of lean and obese Zucker rats treated with vehicle (VEH), 1.5 mg / kg of compound or 3.0 mg / kg of compound 2. It is a graph. Treated with vehicle (VEH), 1.5 mg / kg of compound or 3.0 mg / kg of compound 2 during the experimental treatment period (test days 10-17) and drug holiday (test day 24). It is a bar graph of the weight change percentage with time which shows the percentage weight change of the lean Zucker rat and the obese Zucker rat. Rats fed a standard solid diet and rats fed a high-fat diet treated with vehicle, 1.5 mg / kg compound or 3.0 mg / kg compound 2 at the baseline, treatment, and washout stages of the study. It is a graph of the cumulative food intake with time which shows the cumulative food intake. Rats fed a standard solid diet and rats fed a high-fat diet treated with vehicle, 1.5 mg / kg compound or 3.0 mg / kg compound 2 at the baseline, treatment, and washout stages of the study. It is a graph of the average body weight with time which shows the average body weight. Percentages of rats fed a standard solid diet and rats fed a high-fat diet treated with vehicle, 1.5 mg / kg of Compound or 3.0 mg / kg of Compound 2. Percentage of weight changes over time. It is a bar graph of. 12A. It is a graph of Nrf2 expression under various conditions including a knockdown condition. 12B. It is a graph of NQO1 expression under various conditions including knockdown conditions. It is a graph of EPHX1 expression under various conditions including a knockdown condition. 13A. FIG. 6 is a bar graph of multiple changes in COX-2 mRNA expression showing that Compound 1 has no effect on COX-2 transcription. COX-2 mRNA expression analysis of untreated HeLa cells (control) by qRT-PCR was compared to HeLa cells treated with 10 μM compound 1, 20 ng / ml TNFα, or 10 μM compound 1 + 20 ng / ml TNFα. .. 13B. FIG. 5 is a graph of COX-2 protein expression intensity showing that Compound 1 inhibits TNFα-induced COX-2 protein expression. Images of cells treated with DMSO, 20 ng / mL TNFα, or compound 1 + 20 ng / mL TNFα, showing localization of various inflammation-related CRM1 cargo proteins. Images of cells treated with DMSO, 20 ng / mL TNFα, or compound 1 + 20 ng / mL TNFα showing localization of IκB, NFκB, NRF2, PPARγ, and RXRα. Platform as a function of time in MWM trials showing the effect of sham treatment, control treatment, progesterone treatment, and various doses of Compound 1 on the delay time of mice to reach the platform during the acquisition phase of the MWM trial. It is a graph of the delay time until reaching (the data represents the average value ± SEM). It is a graph of the cytokine concentration which shows the concentration of some cytokines in rat plasma. It is a photograph of the whole brain of an animal treated with sham, CCI + vehicle (control), or CCI + Compound 1 (6 mg / kg) showing the results of a qualitative visual examination of the entire brain before vibratome excision. Examination suggested that none of the Sham animals (0 of 4) showed damage to the dorsal-medial cortex tissue. In stark contrast, all four CCI controls showed severe bilateral injury confined to this area of the cortex. CCI animals treated with Compound 1 showed moderate to minimal damage. In particular, even the brain showing the most severe injury in the compound 1 group was not so extreme as compared with all the brains in the CCI control group. Low magnification micrographs of NeuN-labeled dorsal and ventral cortex regions in sham, CCI + vehicle-treated (control), and CCI + Compound 1-treated (KPT) animals. Immunofluorescent labeled micrographs of rat IgG and TNFα in sham, CCI + vehicle treated (control), and CCI + compound 1 treated (KPT) animals. FIG. 5 is a graph of clinical scores as a function of time showing arthritis scores in unsensitized female Lewis rats, control arthritis female Lewis rats, or arthritis female Lewis rats treated with Compound 2. In a graph of arthritis as a function of time, showing joint swelling as assessed on a scale of 0-4 in unsensitized female Lewis rats, control arthritis female Lewis rats, or arthritic female Lewis rats treated with Compound 2. is there. FIG. 5 is a graph of tarsal bone mineral density (BMD) in unsensitized female Lewis rats, control arthritic female Lewis rats, or arthritic female Lewis rats treated with Compound 2. Visualization by three-dimensional micro-CT imaging of the hind paw of unsensitized female Lewis rat, control arthritic female Lewis rat, or arthritic female Lewis rat treated with Compound 2. 17C. CIA test No. IL-1β concentration in synovial fluid collected from group A (unsensitized), group B (model), and group C (5 mg / kg daily compound 2) rats on days 21 and 27 of 2. It is a graph of IL-1β concentration in synovial fluid as a function of time. 17D. CIA test No. IL-6 concentration in synovial fluid collected from group A (unsensitized), group B (model), and group C (5 mg / kg daily compound 2) rats on days 21 and 27 of 2. It is a graph of IL-6 concentration in synovial fluid as a function of time. 17E. CIA test No. Concentrations of MCP-1 in synovial fluid collected from group A (unsensitized), group B (model), and group C (5 mg / kg daily compound 2) rats on days 21 and 27 of 2. It is a graph of the MCP-1 concentration in synovial fluid as a function of time. 17F. CIA test No. Shows the CRP concentrations in synovial fluid collected from group A (unsensitized), group B (model), and group C (5 mg / kg daily compound 2) rats on days 21 and 27 of 2. , Is a graph of CRP concentration in synovial fluid as a function of time. CIA test No. IL-1β in rat serum samples taken from group A (unsensitized), group B (model), and group C (5 mg / kg daily compound 2) rats on days 15 and 27 of 2. FIG. 6 is a graph of serum IL-1β concentration as a function of time, indicating concentration. It is a schematic diagram of the MOG-induced EAE mouse model in the female mouse described in this specification. FIG. 5 is a graph of clinical scores as a function of test day showing the effects of vehicle treatment, dexamethasone treatment, and compound 1 treatment on the clinical scores of female mice in the MOG-induced EAE mouse models described herein. FIG. 6 is a photograph of a wound locally or systemically treated with Compound 1 or its appropriate vehicle, showing the results of a wound morphological evaluation performed 5 days after wound formation.

The first embodiment of the compound of the invention is of formula I:
Compound, or a pharmaceutically acceptable salt thereof,
During the ceremony
R ¹ is selected from hydrogen and C _{1 to} C ₄ alkyl;
R ² is selected from O and S;
R ^{3 _{is, -N (R 4) - (}} C 3 ~C 6 cycloalkyl), _- C 1 -C ₆ alkyl, - _(C 0 -C ₄ alkylene) - heterocyclyl, and - _(C 0 -C ₄ alkylene ) - it is selected from heteroaryl, (wherein, all alkyl, alkylene, heterocyclyl or heteroaryl moiety, the R ³ is substituted independently optionally);
R ⁴ is selected from hydrogen and C _{1 to} C ₄ alkyl.

In the first aspect of the first embodiment, R ¹ is selected from hydrogen and methyl. The values of the remaining variables are described in the first embodiment.

In the second aspect of the ^first embodiment, R ¹ is hydrogen. The values of the remaining variables are described in the first embodiment.

In the third aspect of the first embodiment, R ² is O. The values of the remaining variables are described in the first embodiment, or the first or second aspect thereof.

In the fourth aspect of the first embodiment, R ² is S. The values of the remaining variables are described in the first embodiment, or first to third aspects thereof.

In a fifth aspect of the first embodiment, R ⁴ is hydrogen.

In the sixth aspect of the first embodiment, R ³ is -N (R ⁴ )-(C _{3 to} C ₆ cycloalkyl), -C _{3 to} C ₆ alkyl,-(C _{0 to} C ₁ alkylene). Selected from − heterocyclyl, and − (C _{0 to} C ₁ alkylene) -heteroaryl, any alkyl or alkylene moiety of R ³ is optionally −N (R ⁵ ) ₂ (in the formula, each R ⁵ is substituted with independently selected from hydrogen and C ₁ -C ₄ alkyl); any heterocyclyl, and heteroaryl portion of R ³ is comprises at least one nitrogen atom in the ring; any of R ³ heterocyclyl, and heteroaryl moieties are optionally substituted with a C ₁ -C ₄ alkyl. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

In the seventh aspect of the first embodiment, R ³ is -C (CH ₃ ) ₃ , -CH (NH ₂ ) -CH (CH ₃ ) ₂ , -NH-cyclopropyl,-(CH ₂ ) _{0. to 1} - it is selected morpholin-4-yl, and methyl pyrazolyl - pyrazinyl, piperidinyl, hydroxy piperidinylmethyl, N- methylpiperidinyl, -CH _2. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

In the eighth aspect of the first embodiment, R ³ is -C (CH ₃ ) ₃ , -CH (NH ₂ ) -CH (CH ₃ ) ₂ , -NH-cyclopropyl,-(CH ₂ ) _{0. ~ 1} - pyrazin-2-yl, piperidin-3-yl, -CH ₂ - is selected from morpholin-4-yl, and 5-methyl -1-H- pyrazol-4-yl. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

In the ninth aspect of the first embodiment, R ³ is -C (CH ₃ ) ₃ , -NH-cyclopropyl, -CH ₂ -pyrazine-2-yl, -pyrazine-2-yl, -CH ₂ -Morpholine-4-yl and 5-methyl-1-H-pyrazole-4-yl are selected. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

A second embodiment is compound (I) of the formula, or a pharmaceutically acceptable salt thereof, wherein.
R ^{3 _{is, -N (R 4) - (}} C 3 ~C 6 cycloalkyl), _- C 3 -C ₆ alkyl, - _(C 0 -C ₁ alkylene) - heterocyclyl, and - _(C 0 -C ₁ alkylene ) -Selected from heteroaryl,
During the ceremony
Any alkyl or alkylene moiety of R ³ may optionally, -N ^(R _{5) 2} (wherein each ^{R 5} is independently selected from hydrogen and _C 1 -C ₄ alkyl) substituted with ,;
Any heterocyclyl, and heteroaryl portion of R ³ is comprises at least one nitrogen atom in the ring;
Any heterocyclyl, and heteroaryl portion of R ³ may optionally _be substituted by C 1 -C ₄ alkyl.

In the first aspect of the second embodiment, R ³ is -C (CH ₃ ) ₃ , -CH (NH ₂ ) -CH (CH ₃ ) ₂ , -NH-cyclopropyl,-(CH ₂ ) _{0. to 1} - it is selected morpholin-4-yl, and methyl pyrazolyl - pyrazinyl, piperidinyl, hydroxy piperidinylmethyl, N- methylpiperidinyl, -CH _2.

In the second aspect of the second embodiment, R ³ is -C (CH ₃ ) ₃ , -CH (NH ₂ ) -CH (CH ₃ ) ₂ , -NH-cyclopropyl,-(CH ₂ ) _{0. ~ 1} - pyrazin-2-yl, piperidin-3-yl, -CH ₂ - is selected from morpholin-4-yl, and 5-methyl -1-H- pyrazol-4-yl.

In the third aspect of the second embodiment, R ³ is -C (CH ₃ ) ₃ , -NH-cyclopropyl, -CH ₂ -pyrazine-2-yl, -pyrazine-2-yl, -CH ₂ -Morpholine-4-yl and 5-methyl-1-H-pyrazole-4-yl are selected.

A third embodiment provides a compound of formula I, or a pharmaceutically acceptable salt thereof,
During the ceremony
R ^{3 _{is, -N (R 4) - (}} C 3 ~C 6 cycloalkyl), _- C 3 -C ₆ alkyl, - _(C 0 -C ₁ alkylene) - heterocyclyl, and - _(C 0 -C ₁ alkylene ) -Selected from heteroaryl, in the formula,
Any alkyl or alkylene moiety of any R ³ is optionally and independently substituted with one or more substituents selected from the group consisting of oxo and -N (R ⁵ ) ₂ in the formula. each ^{R 5} is independently selected from hydrogen and _C 1 -C ₄ alkyl;
Any heterocyclyl moiety R ³ contains at least a one nitrogen atom in the ring, optionally substituted with one or more substituents selected from the group consisting of C ₁ -C ₄ alkyl and oxo Be;
Every heteroaryl moiety of R ³ comprises at least one nitrogen atom in the ring and is optionally substituted with one or more C _{1 to} C ₄ alkyl. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

In the first aspect of the ^third embodiment, R ³ is − (C _{0 to} C ₁ alkylene) -heterocyclyl. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

In the second aspect of the ^third embodiment, R ³ is-(C _0- C ₁ alkylene) -heterocyclyl, in which the heterocyclyl is selected from pyrazinyl, piperidinyl, morpholinyl, and pyrazolyl. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

In the third aspect of the third embodiment, R ³ is − (C _{0 to} C ₁ alkylene) -heterocyclyl, and the heterocyclyl is morpholinyl. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

In the fourth aspect of the ^third embodiment, R ³ is-(C ₁ alkylene) -heterocyclyl. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

In a fifth aspect of the ^third embodiment, R ³ is-(C ₁ alkylene) -morpholinyl. The values of the remaining variables are described in the first embodiment, or first to fifth aspects thereof.

Illustrative compounds of formula I are listed in Table 1.

In some embodiments, the compounds of the invention are selected from any one of compounds 1-11. In one aspect of these embodiments, the compound is selected from any one of compounds 1, 2, 3, 4, 8, and 11.

Pharmacokinetics (PK) is playing an increasingly important role in drug discovery research and development. Pharmacokinetics is a quantitative study of changes in drug absorption, distribution, metabolism and / or excretion over time. When a drug is administered, it is rapidly distributed from the site of administration to the systemic blood circulation. One measure of the degree of distribution of the therapeutic agent is the area under the plasma concentration-time curve (AUC), calculated to the final measured concentration (AUC _t ) and extrapolated to infinite time (AUC _Inf ). Therefore, AUC is frequently used to quantify drug exposure.

In general, the higher the therapeutic agent exposure, the greater the effect of the agent. However, high therapeutic drug exposure can also have detrimental effects on certain tissues, such as the brain. The blood-brain barrier (BBB), a protective network of tight junctions between endothelial cells, limits the diffusion of hydrophilic and / or large molecules, while drugs with high AUC still have BBB and / or cerebrospinal fluid. Can be transmitted through. Such permeation can often lead to unwanted and unwanted side effects. Current drug discovery efforts, in part, aim to successfully balance maximization of drug exposure (ie, AUC) with minimization of brain permeability.

The brain-plasma (B: P) ratio is one such (once) method of quantifying the relative distribution of therapeutic agents in circulating brain tissue. Such a ratio provides an indicator of brain permeability of a given therapeutic agent. High brain: plasma ratios are preferred when targeting diseases localized in the central nervous system (CNS), including the brain and cerebrospinal fluid. However, lower brain-to-plasma ratios are generally preferred for non-CNS therapeutics in order to minimize brain permeability and avoid possible side effects. Therefore, a low brain-to-plasma ratio is preferred to avoid the accumulation of unwanted therapeutic agents in the brain and CNS tissues.

As described in more detail in the Examples section, the compounds of the present invention were filed on March 5, 2011 and published as US Patent Application Publication No. 2009/0275607 on November 10, 2011. Higher AUC and / or lower B: P compared to other nuclear transport inhibitors, such as those disclosed in U.S. Patent Application No. 13 / 041,377, Common Owner. Shown. In some embodiments, the invention provides a compound of formula I, which, when orally administered to mice at 10 mg / kg, has a nuclear export activity of <1 μM (less than 1 μM), greater than about 3500. It has an AUC _Inf ; and a B: P of less than about 2.5.

The novel properties of the present invention will become apparent to those skilled in the art by analyzing the detailed description of the invention below. However, from the detailed description of the invention and the subsequent claims, various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art, and thus the detailed description and specific practice of the reported invention. It should be understood that the examples represent particular embodiments of the present invention, while provided for purposes of illustration only.

Compounds and Definitions The compounds of the invention include those outlined above and further illustrated by the classes, subclasses, and classes disclosed herein. Unless otherwise specified, the following definitions shall apply in the usage of this specification. For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition. In addition, the general principles of organic chemistry are incorporated herein by reference in their entirety, "Organic Chemistry", Thomas Sorrel, Universality Science Books, Sausalito: 1999, and "March's Chemistry". Edition, Ed. : Smith, M.M. B. and March, J. et al. , John Wiley & Sons, New York: 2001.

Unless otherwise specified herein, the nomenclature used herein is the Nomenclature of Organic Chemistry, Sections, which is incorporated herein by reference for exemplary chemical structure names and chemical structure nomenclatures. The examples and rules described in A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979 are generally followed. Optionally, the compound name is given to the Chemical Naming Program ACD / ChemSketch, Version 5.09 / September 20011, Advanced Chemistry Development, Inc. , Toronto, Canada may be used to generate.

The compounds of the present invention are described (eg, as described in EL Eliel and SH Wilen, Stereo-chemistry of Carbon Compounds, John Willey & Sons, New York, 1994, pages 1119-1190). It has a center, a chiral axis, and a chiral plane and may exist as a racemate, a mixture of racemates, and as individual diastereomers or enantiomers, all possible isomers, including optical isomers. And mixtures thereof are included in the present invention.

The term "halo" or "halogen", as used herein, means halogen, including, but not limited to, both radioactive and non-radioactive forms of fluoro, chloro, bromo, iodo and the like.

The term "alkyl" as used herein is a linear or branched saturated monovalent hydrocarbon free, typically C _{1 to} C ₁₂ , preferably C _{1 to} C ₆ , unless otherwise noted. Means a radical. Thus, "C _1- C ₆ alkyl" means a linear or branched saturated monovalent hydrocarbon radical having 1 to 6 (eg, 1, 2, 3, 4, 5 or 6) carbon atoms. To do. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, and t-butyl.

Substituents and substitution patterns on the compounds of the present invention have been selected by those skilled in the art to provide compounds that are chemically stable and can be readily synthesized by techniques known in the art and by the methods described below. Understood as gaining. In general, the term "substituted" means that one or more hydrogens in a specified portion are substituted with the appropriate substituents, regardless of whether the term "optionally" precedes it. Means. Unless otherwise stated, an "arbitrarily substituted group" can have an appropriate substituent at each substitutable position of the group, with two or more positions within any given structure. Substituents can be the same or different at each position if they may be substituted with two or more substituents selected from a particular group. Alternatively, the "optionally substituted group" can be unsubstituted.

The combination of substituents envisioned by the present invention preferably results in the formation of stable or chemically feasible compounds. If the substituents themselves are substituted by more than one group, it is understood that these multiple groups can be on the same or different carbon atoms, provided that a stable structure is obtained. .. The term "stable" is used herein because of their generation, detection, and, in certain embodiments, their recovery, purification, and one or more of the purposes disclosed herein. Refers to a compound that does not substantially denature when placed under conditions that allow its use.

Appropriate monovalent substituents on substitutable carbon atoms of "optionally substituted groups" are independently halogen;-(CH ₂ ) _0-4 R °;-(CH ₂ ) _{0. ~ 4} OR °; −O (CH ₂ ) _{0 to 4} R °, −O− (CH ₂ ) _{0 to 4} C (O) OR °; − (CH ₂ ) _{0 to 4} CH (OR °) ₂ ; − (CH ₂ ) _{0 to 4} SR °; may be replaced by R °-(CH ₂ ) _{0 to 4} Ph; may be replaced by R °-(CH ₂ ) _{0 to 4} O (CH ₂ ) _{0 ~ 1} Ph; may be substituted with R ° −CH = CHPh; may be substituted with R ° − (CH ₂ ) _{0 to 4} O (CH ₂ ) _{0 to 1} − pyridyl; −NO ₂ ; −CN -N ₃ ;-(CH ₂ ) _0-4 N (R °) ₂ ;-(CH ₂ ) _0-4 N (R °) C (O) R °; -N (R °) C (S) R °;-(CH ₂ ) _0-4 N (R °) C (O) NR ° ₂ ;-N (R °) C (S) NR ° ₂ ;-(CH ₂ ) _0-4 N (R °) ) C (O) OR °; -N (R °) N (R °) C (O) R °; -N (R °) N (R °) C (O) NR ° ₂ ; -N (R °) ) N (R °) C (O) OR °;-(CH ₂ ) _0-4 C (O) R °;-C (S) R °;-(CH ₂ ) _0-4 C (O) OR ° -(CH ₂ ) _0-4 C (O) SR °;-(CH ₂ ) _0-4 C (O) OSiR ° ₃ ;-(CH ₂ ) _0-4 OC (O) R °; -OC ( O) (CH ₂ ) _0-4 SR-, SC (S) SR °;-(CH ₂ ) _0-4 SC (O) R °;-(CH ₂ ) _0-4 C (O) NR ° ₂ ; -C (S) NR ° ₂ ; -C (S) SR °; -SC (S) SR °,-(CH ₂ ) _0-4 OC (O) NR ° ₂ ; -C (O) N (OR ° ) R °; -C (O) C (O) R °; -C (O) CH ₂ C (O) R °; -C (NOR °) R °;-(CH ₂ ) _0-4 SSR °; _{- (CH 2) 0~4 S (} O) 2 R ° ;-( CH 2) 0~4 S (O) 2 OR ° ;-( CH 2) 0~4 OS (O) 2 R °; -S (O) ₂ NR ° ₂ ;-(CH ₂ ) _0-4 S (O) R °; -N (R °) S (O) ₂ NR ° ₂ ; -N (R °) S (O) ₂ R °; -N (OR °) R °; -C (NH) NR ° ₂ ; -P (O) ₂ R °; -P (O) ) R ° ₂ ; -OP (O) R ° ₂ ; -OP (O) (OR °) ₂ ; SiR ° ₃ ;-(C _1-4 linear or branched alkylene) ON (R °) ₂ ; Or-(C _1-4 linear or branched alkylene) C (O) ON (R °) ₂ , where each R ° may be substituted as defined below. Independently, hydrogen, C _1-6 aliphatic, -CH ₂ Ph, -O (CH ₂ ) _0-1 Ph, -CH _2- (5- to 6-membered heteroaryl ring), or nitrogen, oxygen, and sulfur. 5-6 membered saturated, partially unsaturated, or aryl rings with 0 to 4 heteroatoms selected independently of, or two independent R ° without being disturbed by the above definition. The presence, together with their intervening atoms, is a 3- to 12-membered saturated, partially unsaturated, or aryl monocycle with 0 to 4 heteroatoms selected independently of nitrogen, oxygen, and sulfur. Alternatively, it may form a bicycle, which may be replaced as defined below.

Appropriate monovalent substituents on R ° (or the ring formed by combining the presence of two independent R ° with their intervening atoms) are independently halogen,-(CH ₂ ) _{0. ~ 2} R ^● ,-(haloR ^● ),-(CH ₂ ) _0-2 OH,-(CH ₂ ) _0-2 OR ^● ,-(CH ₂ ) _0-2 CH (OR ^● ) ₂ ; -O ( Halo R ^● ), -CN, -N ₃ ,-(CH ₂ ) _{0 to 2} C (O) R ^● ,-(CH ₂ ) _{0 to 2} C (O) OH,-(CH ₂ ) _{0 to 2} C (O) OR ^● ,-(CH ₂ ) _0-2 SR ^● ,-(CH ₂ ) _0-2 SH,-(CH ₂ ) _0-2 NH ₂ ,-(CH ₂ ) _0-2 NHR ^● ,- (CH ₂ ) _{0 to 2} NR ^● ₂ , -NO ₂ , -SiR ^● ₃ , -OSiR ^● ₃ , -C (O) SR ^● ,-(C _1-4 linear or branched alkylene) C (O) OR ^● or −SSR ^● , in which each R ^● is unsubstituted, or if “halo” precedes it, it is replaced with only one or more halogens, C _1-4 aliphatic, − CH ₂ Ph, -O (CH ₂ ) _{0 to 1} Ph, or 5 to 6 member saturated, partially unsaturated, or having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Selected independently of the aryl ring. Suitable divalent substituents on saturated carbon atoms at R ° include = O and = S.

Suitable divalent substituents on the saturated carbon atom of the "optionally substituted group" include = O, = S, = NNR ^* ₂ , = NNHC (O) R ^* , = NNHC (O). OR ^* , = NNHS (O) ₂ R ^* , = NR ^* , = NOR ^* , -O (C (R ^* ₂ )) _2-3 O-, and -S (C (R ^* ₂ )) _2-3 The presence of each independent R ^* in the formula, including S-, is selected independently of hydrogen, C _1-6 aliphatics which may be substituted as defined below, or nitrogen, oxygen, and sulfur. It is selected from unsubstituted 5-6 membered saturated, partially unsaturated or aryl rings having 0 to 4 heteroatoms. Suitable divalent substituents that attach to adjacent substitutable carbons of "optionally substituted" groups include -O (CR ^* ₂ ) _2-3 O-, each of which is included in the formula. The presence of an independent R ^* contains 0-4 heteroatoms independently selected from hydrogen, C _1-6 aliphatics, which may be substituted as defined below, or nitrogen, oxygen, and sulfur. It is selected from unsaturated 5- to 6-membered saturated, partially unsaturated or aryl rings having.

Suitable substituents on the aliphatic group of R ^* include halogen, -R ^● ,-(haloR ^● ), -OH, -OR ^● , -O (haloR ^● ), -CN, -C (O) OH. , -C (O) OR ^● , -NH ₂ , -NHR ^● , -NR ^● ₂ , and -NO _2, where each R ^● is unsubstituted or preceded by "halo". In the case of being replaced with only one or more halogens, independently from C _1-4 aliphatic, -CH ₂ Ph, -O (CH ₂ ) _0-1 Ph, or nitrogen, oxygen, and sulfur. It is a 5- to 6-membered saturated, partially unsaturated, or aryl ring with 0 to 4 heteroatoms selected independently.

Suitable substituents on substitutable nitrogen for "optionally substituted groups" are -R ^† , -NR ^† ₂ , -C (O) R ^† , -C (O) OR ^† ,-. C (O) C (O) R ^† , -C (O) CH ₂ C (O) R ^† , -S (O) ₂ R ^† , -S (O) ₂ NR ^† ₂ , -C (S) NR ^† ₂ , -C (NH) NR ^† ₂ , and -N (R ^† ) S (O) ₂ R ^† are mentioned; in the equation, each R ^* is independently defined as hydrogen, as defined below. C _1-6 aliphatic, unsubstituted-OPh, which may be substituted with, or unsubstituted 5-6 membered saturation with 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. , Selected from partially unsaturated or aryl rings, or without being disturbed by the above definition, the presence of two independent R ^† , together with their intervening atoms, is independent of nitrogen, oxygen, and sulfur. To form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl monocycle or bicycle having 0 to 4 heteroatoms selected.

Suitable substituents on the aliphatic group of R ^† are independently halogen, -R ^● ,-(haloR ^● ), -OH, -OR ^● , -O (haloR ^● ), -CN, -C ( O) OH, -C (O) OR ^● , -NH ₂ , -NHR ^● , -NR ^● ₂ , or -NO ₂ , and in the formula, each R ^● is unsubstituted or "halo" is If preceded, substituted with only one or more halogens, independently, C _1-4 aliphatic, -CH ₂ Ph, -O (CH ₂ ) _0-1 Ph, or nitrogen, oxygen, and. A 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms selected independently of the sulfur.

Preferred substituents on the heteroaryl are -OH, -SH, nitro, halogen, amino, cyano, C _{1 to} C ₁₂ alkyl, C _{2 to} C ₁₂ alkenyl, C _{2 to} C ₁₂ alkynyl, C _{1 to} C ₁₂ alkoxy. , C _{1 to} C ₁₂ haloalkyl, C _{1 to} C ₁₂ haloalkoxy, and C _{1 to} C ₁₂ alkyl sulfanyl. Preferred substituents on alkyl, alkylene, and heterocyclyl include preferred substituents on heteroaryl and oxo. In one embodiment, the alkyl, alkylene, the substituents on a heterocyclyl or heteroaryl is an amino group having the formula -N ^(R _{5) 2,} each ^{R 5} is, independently from hydrogen and _C 1 -C ₄ alkyl Is selected.

Substituents on alkyl, alkylene, heterocyclyl, and heteroaryl are -OH, -SH, nitro, halogen, amino, cyano, C _1- C ₁₂ alkyl, C _2- C ₁₂ alkenyl, C _2- C. _{It can be selected from 12} alkynyl groups, C _{1 to} C ₁₂ alkoxy, C _{1 to} C ₁₂ haloalkyl, C _{1 to} C ₁₂ haloalkoxy, and C _{1 to} C ₁₂ alkyl sulfanyl. In one embodiment, the substituent has the formula, an amino group having -N ^(R _{5) 2,} wherein each ^{R 5} is independently selected from hydrogen and _C 1 -C ₄ alkyl.

The term "cycloalkyl" as used herein means a saturated cyclic hydrocarbon, a compound in which all ring atoms are carbon. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. In some embodiments, the cycloalkyl is optionally -OH, -SH, halogen, amino, nitro, cyano, C _{1 to} C ₁₂ alkyl, C _{2 to} C ₁₂ alkoxy or C _{2 to} C ₁₂ alkynyl. It can be substituted with one or more substituents selected from groups, C _{1 to} C ₁₂ alkoxy, C _{1 to} C ₁₂ haloalkyl, and C _{1 to} C ₁₂ haloalkoxy.

The term "heteroaryl" as used herein refers to an aromatic group containing one or more heteroatoms (O, S, or N). The heteroaryl group can be monocyclic or polycyclic, for example a monocyclic heteroaryl ring fused to one or more carbocyclic aromatic groups or other monocyclic heteroaryl groups. The heteroaryl group of the present invention may also include a ring system substituted with one or more oxo moieties. Examples of heteroaryl groups are pyridinyl, pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, frills, thienyl, isooxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzoimidazolyl. Benzofuranyl, cinnolinyl, indazolyl, indolidinyl, phthalazinyl, pyridadinyl, triazinyl, isoindrill, prynyl, oxadiazolyl, thiazolyl, thiadiazolyl, frazanyl, benzofrazanyl, benzothiophenyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl Examples include, but are not limited to, dihydroquinolyl, tetrahydroquinolyl, dihydroisoquinolyl, tetrahydroisoquinolyl, benzofuryl, flopyridinyl, pyrolopyrimidinyl, and azaine drill.

The heteroaryl groups described above may be C-adherent or N-adherent (where possible). For example, the group derived from pyrrole may be pyrrole-1-yl (N-adhesive) or pyrrole-3-yl (C-adhesive).

"Heterocyclyl" means a 4- to 13-membered saturated or unsaturated aliphatic ring containing 1, 2, 3, 4 or 5 heteroatoms selected independently of N, O or S. .. If one heteroatom is S, it can optionally be mono- or dioxygenated (ie-S (O)-or -S (O) _2- ). Heterocyclyls can be monocyclic, fused bicyclic, crosslinked bicyclic, spirobicyclic or polycyclic.

"Oxo" means = O.

As used herein, the term "alkenyl" means a saturated straight chain or branched acyclic hydrocarbon having 2-12 carbon atoms and at least one carbon-carbon double bond. To do. The alkenyl group may optionally be substituted with one or more substituents.

As used herein, the term "alkynyl" means a saturated linear or branched acyclic hydrocarbon having 2-12 carbon atoms and at least one carbon-carbon triple bond. .. The alkynyl group may optionally be substituted with one or more substituents.

As used herein, the term "alkylene" refers to an alkyl group that has two attachment points to the rest of the compound. Non-limiting examples of the alkylene group include methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH (CH3)-) and the like. The alkylene group may optionally be substituted with one or more substituents.

The term "haloalkyl" includes, in usage herein, an alkyl substituted with one or more F, Cl, Br, or I, the alkyl being defined above.

The term "alkoxy", as used herein, means an "alkyl-O-" group, where alkyl is defined above. Examples of alkoxy groups include methoxy or ethoxy groups.

As used herein, the term "pharmaceutically acceptable salt" is used with human and lower animal tissues, within sound medical judgment, without undue toxicity, irritation, allergic responses, etc. A salt that is suitable for contact use and has a reasonably balanced benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. Is J. Pharmaceutical Sciences, 1977, 66, 1-19, describe in detail pharmaceutically acceptable salts. Pharmaceutically acceptable salts of the compounds of the invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are by inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or by acetic acid, trifluoroacetic acid (2,2,2-). Formed by organic acids such as trifluoroacetic acid), oxalic acid, maleic acid, tartrate acid, citric acid, succinic acid or malonic acid, or by other methods used in the art such as ion exchange. It is a salt of an amino group. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, asparagate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, succinate. , Drosophila sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate Salt, heptaneate, hexaneate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonic acid Salt, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate , Phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, trifluoroacetic acid (2,2,2-trifluoro) Acetic acid), undecanoate, valerate and the like.

Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and ^N + _{(C 1 to 4 alkyl) 4} salts. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Additional pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryls. Examples include amine cations formed using counterions such as sulfonates.

Unless otherwise noted, the structures described herein are all structures, such as the R and S configurations of each asymmetric center, the Z and E double bond isomers, and the Z and E conformational isomers. It is also intended to include isomer (eg, enantiomer, diastereomeric, and geometric (or stereostructural)) forms of. Thus, single steric chemical isomers of the compounds, as well as enantiomers, diastereomers, and geometric (or three-dimensional) mixtures are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. In addition, unless otherwise stated, the structures described herein are intended to include compounds that differ only in the presence of one or more isotope enriched atoms. For example, compounds having this structure comprising substitution of hydrogen with deuterium or tritium, or substitution of carbon with ¹³ C enriched or ¹⁴ C enriched carbon are within the scope of the present invention. Such compounds are useful, for example, as analytical tools, as probes for biological assays, or as therapeutic agents according to the present invention.

The term "pharmaceutically acceptable salt" means either an acid addition salt or a base addition salt that is suitable for the patient's treatment.

In some embodiments, exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and acids such as sodium monohydrogen orthorate, and potassium hydrogen sulfate. Metal salts include, but are not limited to. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids. Examples of such acids include acetic acid, trifluoroacetic acid (2,2,2-trifluoroacetic acid), glycolic acid, lactic acid, pyruvate, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, Tartrate acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, 2-phenoxybenzoic acid, p-toluenesulfonic acid and methanesulfonic acid and 2-hydroxyethane Other sulfonic acids such as sulfonic acids. Either a monoate or a diate can be formed, such salts can be present in either hydrated, solvated or substantially anhydrous forms. In general, acid addition salts of these compounds are more soluble in water and various hydrophilic organic solvents as compared to their free base form and generally exhibit a higher melting point. For laboratory use or for subsequent conversion to pharmaceutically acceptable acid addition salts, for example in the isolation of compounds of formula I, other pharmaceutically acceptable, such as oxalate. You may use non-salt.

A "pharmaceutically acceptable basic addition salt" is any non-toxic organic or inorganic base addition salt of the acid compound represented by Formula I, or an intermediate thereof. Illustrative inorganic bases that form suitable salts include, but are not limited to, hydroxides of lithium, sodium, potassium, calcium, magnesium or barium. Illustrative organic bases that form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine, and picoline, or ammonia. If there are ester functional groups elsewhere in the molecule, it may be important to select the appropriate salt so that it is not hydrolyzed. Appropriate salt selection criteria are known to those of skill in the art.

The acid addition salts of the compounds of formula I are most appropriately produced from pharmaceutically acceptable acids, such as inorganic acids such as hydrogen chloride, sulfuric acid or phosphoric acid, and eg succinic acid, maleic acid, acetic acid, tri. Examples include those formed by organic acids such as fluoroacetic acid or fumaric acid. For laboratory use or for subsequent conversion to pharmaceutically acceptable acid addition salts, for example in the isolation of compounds of formula I, other pharmaceutically acceptable, such as oxalate. You may use non-salt. Base addition salts (such as sodium, potassium, and ammonium salts), solvate compounds, and hydrates of the compounds of the invention are also included within the scope of the invention. Converting a given compound salt to the desired compound salt is accomplished by applying standard techniques well known to those skilled in the art.

The term "three isomers" is a general term that refers to all isomers of individual molecules that differ only in the orientation of their atoms in space. This includes enantiomers (enantiomers), geometric (cis / trans) isomers, and isomers of compounds that have two or more chiral centers that are not diastereomers of each other.

The term "treat" or "treatment", on a temporary or permanent basis, relieves symptoms, eliminates the cause of symptoms, prevents the above-mentioned disorders or medical conditions or manifestations, or Means to delay.

The term "therapeutically effective amount" means the amount of a compound that is effective in treating or reducing the severity of one or more symptoms of a disease or condition.

When referring to the elements disclosed herein, the articles "a", "an", "the", and "said" are intended to mean that there are one or more elements. Will be done. The terms "contain", "have", and "beginning" are intended to be unconstrained, meaning that there may be additional elements in addition to the elements listed.

Use, Formulation, and Administration Pharmaceutically Acceptable Compositions According to another embodiment, the invention is the compound of the invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or. A composition comprising a vehicle is provided. The amount of compound in the composition of the invention is an amount effective in inhibiting CRM1 measurable in a biological sample or in a patient. In certain embodiments, the compositions of the invention are formulated for administration to a patient in need of such a composition. The term "patient" as used herein means an animal. In some embodiments, the animal is a mammal. In certain embodiments, the patient is a veterinary patient (ie, a non-human mammalian patient). In some embodiments, the patient is a dog. In another embodiment the patient is human.

The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that does not disrupt the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the present invention include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, phosphates and the like. Buffers, glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts, colloids Examples include, but are limited to, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, and wool fat. It is not something that is done.

The compositions of the present invention include oral, parenteral administration (subcutaneous, intramuscular, intravenous, and intradermal) through inhalation spray, topical, rectal, intranasal, oral, vaginal, or implantable reservoir. ) May be done. In some embodiments, the provided compound or composition can be administered intravenously and / or intraperitoneally.

The term "parenteral" as used herein is subcutaneously, intravenously, intramuscularly, intraocularly, intravitreal, intraarticularly, intrasynovial, sternum, subarachnoid space, intrahepatic, intraperitoneal. Includes intrafocal and intracranial injection or infusion techniques. Preferably, the composition is administered orally, subcutaneously, intraperitoneally or intravenously. The sterile injectable form of the compositions of the invention may be an aqueous or oily suspension. These suspensions may be formulated by techniques known in the art using suitable dispersions or wetting agents and suspending agents. The sterile injectable formulation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a 1,3-butanediol solution. Acceptable vehicles and solvents that may be used are, among other things, water, Ringer's solution, and isotonic sodium chloride solution. In addition to this, sterile non-volatile oils are conventionally used as solvents or suspension media.

The pharmaceutically acceptable compositions of the present invention include, but are not limited to, capsules, tablets, aqueous suspensions, or solutions in any orally acceptable dosage form. It may be administered orally. For tablets used orally, the carriers commonly used include lactose and cornstarch. Smoothing agents such as magnesium stearate are also typically added. Useful diluents for oral administration in capsule form include lactose and dried cornstarch. If an aqueous suspension is required for oral use, the active ingredient is combined with an emulsifying and suspending agent. Certain sweeteners, flavors or colorants may also be added if desired. In some embodiments, the provided oral formulation is formulated for immediate release or sustained / delayed release. In some embodiments, the composition is suitable for buccal or sublingual administration, including tablets, lozenges, and incense tablets. The compounds provided can also be in microencapsulated form.

Alternatively, the pharmaceutically acceptable compositions of the present invention may be administered in the form of suppositories for rectal administration. The pharmaceutically acceptable compositions of the present invention are also topical, especially if the therapeutic target comprises areas or organs readily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. May be administered as a target. Suitable topical formulations are readily prepared for each of these areas or organs.

Topical application for the lower intestine can be achieved with a rectal suppository formulation (see above) or with a suitable enema formulation. Topical transdermal patches may also be used.

For ophthalmic applications, the pharmaceutically acceptable compositions provided may be formulated as a pulverized suspension or in an ointment such as petrolatum.

The pharmaceutically acceptable compositions of the present invention may also be administered by nasal fumes or inhalation.

In some embodiments, the pharmaceutically acceptable compositions of the present invention are formulated for intraperitoneal administration.

The amount of the compound of the invention that may be combined with the carrier material to produce a single dose composition will vary depending on the host being treated and the particular mode of administration. In one embodiment, the provided compositions should be formulated so that the patient receiving these compositions can receive a dose of an inhibitor at a dose of 0.01-100 mg / kg body weight / day. In another embodiment, the dose is about 0.5 to about 100 mg / kg body weight, or 1 mg to 1000 mg / dose every 4 to 120 hours, or according to the requirements of the particular drug. Typically, the pharmaceutical compositions of the present invention are administered about 1 to about 6 times per day.

Specific doses and treatment plans for any particular patient include activity, age, weight, overall health, gender, diet, dosing time, excretion rate, drug combination, attending physician's judgment, and treatment of the particular compound used. It is also understood that it depends on a variety of factors, including the severity of the particular disease. The amount of the compound of the invention in the composition also depends on the particular compound in the composition.

If desired, maintenance doses of the compounds, compositions or combinations of the invention may be administered to improve the patient's condition. Subsequently, the dose and / or frequency of administration may be reduced depending on the symptoms to a level at which the improved condition is retained when the symptoms are alleviated to the desired level. However, patients may require long-term intermittent treatment for the recurrence of any disease symptoms.

Use of Compounds and Pharmaceutically Acceptable Compositions The compounds and compositions described herein are generally useful for inhibiting CRM1 and are therefore associated with CRM1 activity, one or more. Useful for treating disorders. Thus, in certain embodiments, the invention comprises a step of administering to a patient in need thereof a compound of the invention, or a pharmaceutically acceptable composition thereof, a method of treating a CRM1-mediated disease. I will provide a. The compounds and compositions described herein may also be administered to a subject, for example, in vitro or in vitro to cells in culture, or, for example, in vivo, to begin with the following examples herein. Can treat, prevent, and / or diagnose a variety of disorders.

The activity of the compounds utilized as CRM1 inhibitors in the present invention may be assayed in vitro, in vivo or in cell lines. Detailed conditions for assaying compounds utilized as CRM1 inhibitors in the present invention are described in Examples.

As used herein, the term "CRM1-mediated" disorder or medical condition refers to any disease or other harmful medical condition in which CRM1 is known to play a role in the usage herein. means. Thus, another embodiment of the invention relates to treating or reducing the severity of one or more diseases in which CRM1 is known to play a role. In some embodiments, the invention comprises the step of administering to a patient a therapeutically effective amount of a compound described herein, in the subject, p53, p73, p21, pRB, p27, IκB,. Provided are methods for treating diseases associated with the expression or activity of NFκB, c-Abl, FOXO protein, COX-2, or HDAC (histone deacetylase). In another embodiment, the invention treats, or treats, a disease or condition selected from proliferative disorders (eg, cancer), inflammatory disorders, autoimmune disorders, viral infections, ophthalmic disorders or neurodegenerative disorders. With respect to methods of reducing severity, the method comprises the step of administering the compound or composition according to the invention to a patient in need thereof. In a more specific embodiment, the invention relates to a method of treating or reducing the severity of cancer. Specific examples of the above disorders are described in detail below.

Cancers treatable by the compounds of the present invention include hematological malignancies (leukemia, lymphoma, multiple myeloma and myeloma including myeloma and myeloma proliferative syndrome) and solid tumors (prosthesis, breast, Cancers, but not limited to, cancers of the lungs, colons, pancreas, kidneys, ovaries and soft tissues, osteosarcomas, and interstitial tumors. Breast cancers (BC) include basal cell-like breast cancer (BLBC), triple-negative breast cancer (TNBC), and breast cancer that is both BLBC and TNBC. In addition, breast cancer includes invasive or non-invasive ductal or lobular cancer, tubular, medullary, mucous, nipple, sieve cancer, male breast cancer, recurrent or metastatic breast cancer, phyllodes tumor of the breast. And Paget's disease of the nipple.

Inflammatory diseases that can be treated by the compounds of the present invention include multiple sclerosis, rheumatoid arthritis, degenerative joint disease, systemic lupus erythematosus, systemic sclerosis, vasculitis syndrome (small, medium, and large blood vessels), and atherosclerosis. Arteriosclerosis, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, mucinous colitis, ulcerative colitis, gastric inflammation, sepsis, pemphigus and other dermal inflammatory diseases (eczema, atopic dermatitis, contact dermatitis) , Urticaria, urticaria, and skin diseases with acute inflammatory elements, pemphigus, lupus erythematosus, allergic dermatitis, etc.), and urticaria syndrome, but is not limited to.

Viral diseases treatable by the compounds of the present invention include acute febrile pharyngitis, pharyngeal conjunctivitis, epidemic keratoconjunctivitis, infant gastroenteritis, coxsackie infection, infectious mononuclear disease, barkit lymphoma, acute hepatitis, chronic. Hepatitis, liver cirrhosis, hepatocellular carcinoma, primary HSV-1 infections (eg pediatric gingival stomatitis, adult tonsillitis and pharyngitis, keratoconjunctivitis), opaque HSV-1 infections (eg lip herpes and herpes simplex), Primary HSV-2 infection, opaque HSV-2 infection, aseptic meningitis, infectious mononuclear disease, giant cell inclusion disease, Kaposi sarcoma, multicentric Castleman's disease, primary exudate lymphoma , AIDS, influenza, Rye syndrome, rash, post-infectious encephalomyelitis, mumps cold, hyperplastic epithelial lesions (eg, vulgaris, flattening, sole, and anal genital vulgaris, laryngeal papilla, vulgar epidermis development Abnormalities), cervical cancer, herpes flat epithelial cancer, group, pneumonia, bronchitis, sickness, gray myelitis, mad dog disease, influenza-like syndrome, severe bronchitis with pneumonia, wind rash, congenital wind rash, varicella, And herpes zoster, but is not limited to. Viral diseases that can be treated by the compounds of the present invention also include long-term viral infections, including hepatitis B and hepatitis C.

Exemplary ophthalmic disorders include retinal edema (diabetic and non-diabetic retinal edema), wet and dry retinal aged retinal degeneration, aging discoid retinal degeneration, cystic retinal edema, Eyelid edema, retinal edema, diabetic retinopathy, retinal chorioretinosis, angiogenic yellow spot, angiogenic glaucoma, retinal inflammation, irisitis, retinal angiitis, endophthalmitis, total ophthalmitis, metastatic eye inflammation, choroiditis , Retinal pigment epitheliitis, conjunctivitis, ciliitis, embolitis, epimyelitis, optic neuritis, retrobulbar optic neuritis, keratitis, eyelid inflammation, exudative retinal detachment, corneal ulcer, conjunctival Ulcer, chronic monetary keratitis, eye disease associated with hypoxia or ischemia, premature infant retinopathy, proliferative diabetic retinopathy, polypoid choroidal angiopathy, retinal hemangiomas, retinal artery occlusion, retinal vein Obstruction, Coates' disease, familial exudative vitreous retinopathy, pulseless disease (high anxiety), Eel's disease, antiphospholipid antibody syndrome, leukemia retinopathy, hyperviscosity syndrome, macroglobulinemia, interferon retinopathy , Hypertensive retinopathy, radiation retinopathy, corneal epithelial stem cell insufficiency or cataract, but is not limited to.

Neurodegenerative diseases that can be treated with compounds of formula I include, but are limited to, Parkinson's disease, Alzheimer's disease, and Huntington's disease, and amyotrophic lateral sclerosis (ALS / Lou Gehrig's disease). is not.

The compounds and compositions described herein also include dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, pulmonary fibrosis, liver fibrosis, glomerulonephritis, polycystic nephropathy (PKD), It may also be used to treat abnormal tissue growth and fibrotic disorders, including and other renal disorders.

The compounds and compositions described herein may also be used to treat food intake-related disorders such as obesity and bulimia nervosa.

In another embodiment, asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity, emphysema, chronic bronchitis, acute respiratory distress syndrome, and using the compounds or compositions described herein. Allergic and respiratory diseases, including any chronic obstructive pulmonary disease (COPD), may be treated or prevented.

In some embodiments, the CRM1 activity-related disease or condition is: Muscle dystrophy, such as arthritis such as osteoarthritis and rheumatoid arthritis, ankylosing spontilitis, traumatic brain injury, spinal cord injury, septicemia, rheumatic disease, cancer atherosclerosis, type I diabetes, type II diabetes, Leptospilosis renal disease, glaucoma, retinal disease, aging, headache, pain, complex local pain syndrome, cardiac hypertrophy, muscle wasting, catabolic disorders, obesity, fetal growth retardation, hypercholesterolemia, heart Diseases, chronic heart failure, ischemia / reperfusion, stroke, cerebral aneurysms, angina, lung disease, cystic fibrosis, acid-induced lung injury, pulmonary hypertension, asthma, chronic obstructive lung disease, Schegren's syndrome, lungs Vitreous dysplasia, kidney disease, glomerular disease, alcoholic liver disease, intestinal disease, peritoneal endometriosis, dermatosis, sinusitis, mesoderma, non-sweat exoblast dysplasia with immunodeficiency, Bechet Diseases, dyschromia, tuberculosis, asthma, Crohn's disease, colitis, eye allergies, cystitis, Paget's disease, pancreatitis, periodontitis, endometriosis, inflammatory bowel disease, inflammatory lung disease, silica Induced diseases, sleep aspiration, AIDS, HIV-1, autoimmune diseases, antiphospholipid antibody syndrome, scab, scab nephritis, familial Mediterranean fever, hereditary cycle fever syndrome, psychosocial stress disease, neurological disease Physical disease, familial amyloid polyneuropathy, inflammatory neuropathy, Parkinson's disease, multiple sclerosis, Alzheimer's disease, amyotropic lateral sclerosis, Huntington's disease, cataracts, or hearing loss.

In another embodiment, the CRM1 activity-related disease or condition is as follows: Head trauma, vasculitis, inflammatory pain, allergen-induced asthma, non-allergen-induced asthma, glomerular nephritis, ulcerative colitis, necrotizing enteritis, hyperglobulin Demia with periodic fever (HIDS), TNF Receptor-Associated Periodic Syndrome (TRAPS), Cryopyrin-Associated Periodic Syndrome, McCull Wells Syndrome (Vulgaris-Hearing Absence-Amyloidosis), Familial Cold Urditis, Neonatal-Onset Multi-Organ Inflammatory Diseases Periodic Fever (NOMID), Periodic fever-aphthous stomatitis-pharyngitis-tomorrhea (PFAPA syndrome), Blau syndrome, purulent aseptic arthritis, necrotizing pyoderma, acne (PAPA), interleukin-one receptor antagonist deficiency (DIRA), Spider bleeding, multiple cystic kidneys, transplantation, organ transplantation, tissue transplantation, myelodystrophy syndrome, stimulant-induced inflammation, plant irritant-induced inflammation, turkey / ursiol oil-induced inflammation, chemical irritant induction Caused by sexual inflammation, bee sting-induced inflammation, bite-inducing inflammation, sunlight dermatitis, burns, dermatitis, toxicemia, lung injury, acute respiratory distress syndrome, alcoholic hepatitis, or parasite infections Kidney damage.

In a further aspect, the invention treats diseases associated with the expression or activity of p53, p73, p21, pRB, p27, IκB, NFκB, c-Abl, FOXO protein, COX-2 or HDAC in a subject. Provided is the use of a compound of formula I for producing a drug to be produced. In some embodiments, the present invention relates to cancer and / or neoplastic disorders, angiogenesis, autoimmune disorders, inflammatory and / or disorders, epigenetics, hormonal disorders and / or disorders, viral disorders, Provided is the use of a compound of formula I in the manufacture of a drug for treating any of neurodegenerative disorders and / or diseases, wounds, and ophthalmic disorders.

In some embodiments, the invention comprises contacting a biological sample with a pharmaceutically acceptable salt of a compound of formula I, or a pharmaceutically acceptable composition thereof, or administering to a patient. Provided are a method of inhibiting CRM1 in a biological sample comprising.

Neoplastic Disorders The compounds or compositions described herein can be used to treat neoplastic diseases. A "neoplastic disorder" is a disease or disorder characterized by cells capable of autonomous proliferation or replication, such as an abnormal condition or condition characterized by proliferative cell growth. Exemplary neoplastic disorders include cancers such as tumors of prostate, brain, bone, colon, lung, milk, ovary, and liver origin, sarcomas, metastatic disorders such as leukemia, lymphoma, myeloma. And other disorders such as malignant plasma cells Hematopoietic neoplastic disorders, and metastatic tumors. Cancers that are widespread include breast, prostate, colon, lung, liver, and pancreatic cancers. Treatment with compounds can be performed in amounts effective to ameliorate at least one symptom of neoplastic disorders, such as reduced cell proliferation, reduced mass mass.

The disclosed methods are for the prevention and treatment of cancers, including, for example, solid tumors, soft tissue tumors, and their metastasis, as well as Lee Fraumeni syndrome, familial breast-ovarian cancer (BRCA1 or BRCA2). It is useful in familial cancer syndromes such as mutation) syndrome. The disclosed methods are also useful in treating non-solid cancers. Exemplary solid tumors include various organ systems such as lung, milk, lymphatic system, digestive organs (eg colon), and urogenital (eg kidney, urinary epithelium, or testicular tumor) pathways, pharynx, prostate, and. Malignant tumors of the ovary (eg, sarcoma, adenocarcinoma, and cancer) can be mentioned. Exemplary adenocarcinomas include colonic rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, and small intestine cancer.

Examples of cancers described by the National Cancer Institute include: Acute lymphoblastic leukemia, adult; acute lymphoblastic leukemia, childhood; acute myeloid leukemia, adult; adrenal cortex cancer; adrenal cortex cancer, childhood; AIDS-related lymphoma; AIDS-related malignancies; anal Hmm; stellate cell tumor, childhood cerebrum; stellate cell tumor, childhood brain; bile duct cancer, extrahepatic; bladder cancer; bladder cancer, childhood; bone cancer, osteosarcoma / malignant fibrous histocytoma; Brain stem glioma, childhood; brain tumor, adult; brain tumor, brain stem glioma, childhood; brain tumor, cerebellar astrocytes, childhood; brain tumor, encephaloma / malignant glioma, childhood; brain tumor, coat Cytoma, childhood; brain tumor, myelobous cell tumor, childhood; brain tumor, cerebral tent tumor Primitive nerve ectodermal tumor, childhood; brain tumor, visual tract and hypothalamic glioma, childhood; brain tumor, childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenoma / Cartinoid, Childhood; Cartinoid Tumor, Childhood; Cartinoid Tumor, Gastrointestinal; Cancer, Adrenal Cortex; Cancer, Pancreatic islet cells; tract primary cancer; central nervous system lymphoma, primary; cerebellar astrocytes, childhood; encephaloma / malignant glioma, childhood; cervical cancer; childhood cancer; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myelinated proliferative disorder; Clear cell sarcoma of tendon sheath; Colon cancer; Colon-rectal cancer, Childhood; Skin T-cell (CeIl) lymphoma; Endometrial cancer; Upper coat cell tumor, childhood; epithelial cancer, ovary; esophageal cancer; esophageal cancer, childhood; Ewing family tumor; extracranial embryonic cell tumor, childhood; extragonal embryonic cell tumor; extrahepatic bile duct cancer; eye Cancer, intraocular melanoma; eye cancer, retinoblastoma; bile sac cancer; gastric cancer; gastric cancer, childhood; gastrointestinal cartinoid tumor; germ cell tumor, extracranial, childhood; germ cell tumor, extragonadal; Germ cell tumor, ovary; gestational chorionic tumor; glioma, childhood brain stem; glioma, childhood visual tract and hypothalamus; hair cell leukemia; head and neck cancer; hepatocellular (liver) cancer, Adult (primary); hepatocellular (liver) cancer, childhood (primary); Hodgkin lymphoma, adult; Hodgkin lymphoma, childhood; Pregnant Hodgkin lymphoma; hypopharyngeal cancer; hypothalamus and visual tract glioma , Childhood; Intraocular melanoma; Pancreatic islet cell carcinoma (endocrine pancreas); Kaposi sarcoma; Kidney cancer; Laryngeal carcinoma; Laryngeal carcinoma, Childhood; Leukemia, Acute lymphoblastic, Adult; Leukemia, Acute Lymphblastic, childhood; leukemia, acute myeloid, adult; leukemia, acute myeloid, childhood; leukemia, chronic lymphocytic; leukemia, chronic myeloid; leukemia, hair cells; lip and oral cancer; Liver cancer, adult (primary); liver cancer, childhood (primary); lung cancer, non-small cells; lung cancer, small cells; lymphoblastic leukemia, adult acute; lymphoblastic leukemia, childhood acute Lymphocytic leukemia, chronic; lymphoma, AIDS-related; lymphoma, central nervous system (primary); lymphoma, cutaneous T cells (CeIl); Hodgkin lymphoma, adult; Hodgkin lymphoma, childhood; Hodgkin lymphoma, pregnant; non- Hodgkin's lymphoma, adult; non-Hodgkin's lymphoma, childhood; non-Hodgkin's lymphoma, pregnant; lymphoma, primary central nervous system; macroglobulinemia, Waldenström; male breast cancer; malignant sarcoma, adult; malignant sarcoma Tumor, childhood; malignant thoracic adenoma; medullary cell tumor, childhood; melanoma; sarcoma, intraocular; merkel cell carcinoma; mesopharyngeal tumor, malignant; metastatic squamous epithelial cervical cancer of unknown primary origin; multiple Sexual endocrine adenoma syndrome, childhood; multiple myeloma / plasma cell tumor; fungal sarcoma; myelodysplastic syndrome; myeloid leukemia, chronic; myeloid leukemia, childhood acute; myeloma, multiple; myeloid proliferative Disease, chronic; nasal and sinus cancer; nasopharyngeal cancer; nasopharyngeal cancer, childhood; sarcoma; non-Hodgkin's lymphoma, adult; non-Hodgkin's lymphoma, childhood; non-Hodgkin's lymphoma during pregnancy; Small cell lung cancer; oral cancer, childhood; oral and lip cancer; mesopharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer, childhood; ovarian epithelial cancer; ovarian germ cell tumor Low malignant potential tumor of the ovary; Pancreatic cancer; Pancreatic cancer, Childhood; Pancreatic cancer, Pancreatic islet cells; Non-nasal and nasal cancer; Parathyroid cancer; Penis cancer; Brown cell tumor; Pine fruit and Cerebral tent Primitive neuroextodermal tumor, childhood; pituitary tumor; plasma cell tumor / multiple myeloma; pleural sarcoma; pregnancy and breast cancer; pregnancy and Hodgkin's lymphoma; pregnancy and non-Hodgkin's lymphoma; primary center Nervous system lymphoma; primary liver cancer, adults; primary liver cancer, childhood; prostate cancer; rectal cancer; renal cell (kidney) cancer; renal cell cancer, childhood; renal pelvis and urinary tract, Transitional epithelial cancer; retinoblastoma; horizontal print myoma, childhood; salivary adenocarcinoma; salivary adenocarcinoma, childhood; Ewing sarcoma family tumor; sarcoma, capos; sarcoma (osteosarcoma) / malignant fibrous tissue of bone Hematoma; sarcoma, rhombic myoma, childhood; sarcoma, soft tissue, adult; sarcoma, soft tissue, childhood; Cesarly syndrome; skin cancer; skin cancer, childhood; skin cancer (black tumor); Skin cancer, Mercel cells; Small cell lung cancer; Small intestinal cancer; Soft tissue sarcoma, Adults; Soft tissue sarcoma, Childhood; Primary unknown flat Cutaneous cervical cancer, metastatic; gastric cancer; gastric cancer, childhood; cerebral tent Ueprimary nerve ectodermal tumor, childhood; T cell (CeIl) lymphoma, skin; testicular cancer; thyroid tumor, childhood; thoracic adenoma , Malignant; thyroid cancer; thyroid cancer, childhood; metastatic epithelial cancer of the renal pelvis and urinary tract; chorionic tumor, fertility; unknown primary site, cancer, childhood; rare cancer in childhood; urine Canal and renal pelvis, metastatic epithelial cancer; urinary tract cancer; uterine sarcoma; vaginal cancer; visual tract and hypothalamic glioma, childhood; genital cancer; Waldenström macroglobulinemia; and Wilms tumor.

Further exemplary cancers include diffuse large cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).

The aforementioned cancer metastases can also be treated or prevented according to the methods described herein.

Combination Therapy In some embodiments, the compounds described herein are administered with an additional "second" therapeutic agent or treatment. The second therapeutic agent may be selected from any agent typically used in monotherapy to treat the indicated disease or condition. As used herein, the term "co-administered" and related terms refer to the simultaneous or sequential administration of therapeutic agents according to the invention. For example, the compounds of the invention may be administered in separate single dosage forms simultaneously or sequentially, or in combination in a single dosage form, together with another therapeutic agent. Accordingly, the present invention provides a single dosage form comprising a compound of formula I, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

In one embodiment of the invention in which the second therapeutic agent is administered to the subject, the effective amount of the compound of the present invention is less than its effective amount when the second therapeutic agent is not administered. In another embodiment, the effective amount of the second therapeutic agent is less than its effective amount when the compound of the invention is not administered. In this way, the unwanted side effects associated with high doses of any of the agents may be minimized. Other possible benefits (including improved dosing regimens and / or reduced drug costs without limitation) will be apparent to those of skill in the art. Additional agents may be administered separately from the compounds of the invention as part of a multi-dose regimen. Alternatively, these agents may be part of a single dose dosage form mixed with a compound of the invention in a single composition.

Cancer Combination Therapy In some embodiments, the compounds described herein are administered with additional cancer therapeutic agents. Exemplary, additional cancer treatments include, for example, targeted therapies such as chemotherapy, antibody therapy, kinase inhibitors, immunotherapy, and hormone therapy, epigenetic therapy, proteosome therapy, and anti-angiogenic therapy. .. Examples of each of these treatments are provided below. As used herein, the terms "combination", "combined" and related terms refer to the simultaneous or sequential administration of therapeutic agents according to the invention. For example, the compounds of the present invention may be administered in separate single dosage forms simultaneously or sequentially, or in combination in a single dosage form, together with another therapeutic agent. Accordingly, the present invention provides a single dosage form comprising the compounds of the invention, additional therapeutic agents, and pharmaceutically acceptable carriers, adjuvants, or vehicles.

The amount of both the compound of the invention and the additional therapeutic agent that can be combined with the carrier material to produce a single dose dosage form (in a composition comprising an additional therapeutic agent as described above). It varies depending on the host being treated and the particular mode of administration. Preferably, the compositions of the invention should be formulated such that the compounds of the invention in doses of 0.01-100 mg / kg body weight / day can be administered.

Chemotherapy In some embodiments, the compounds described herein are administered with chemotherapy. Chemotherapy is a treatment for cancer with drugs that destroy cancer cells. "Chemotherapy" generally refers to cytotoxic agents that affect rapidly dividing cells, as opposed to targeted therapies. Chemotherapeutic agents interfere with cell division in various possible ways, such as DNA replication or separation of newly formed chromosomes. Most forms of chemotherapy target all rapidly dividing cells and are not cancer cell-specific, but DNA damage cannot be repaired by many cancer cells, whereas normal cells generally repair. From what can be done, some specificity may be obtained.

Examples of chemotherapeutic agents used in cancer therapy include, for example, antimetabolites (eg, folic acid, purine, and pyrimidine derivatives), and alkylating agents (eg, nitrogen mustard, nitrosourea, platinum, alkylsulfonate, hydrazine). , Triazen, aziridine, spindle inhibitors, cytotoxic agents, topoisomerase inhibitors, etc.). Exemplary agonists include acralubicin, actinomycin, alitretinoin, altretamine, aminopterin, aminolevulinicin, amrubicin, amsacrine, anagrelide, arsenic trioxide, asparaginase, atlascentan, verotecan, bexalotene, bendamstin, bleomycin, vortezomib, busulphan. , Camptothecin, capecitabin, carboplatin, carbocon, carmofur, carmustin, selecoxib, chlorambusyl, chlormethin, cisplatin, cladribine, clofarabin, chrysanpastase, cyclophosphamide, citarabin, dacarbadine, dactinomycin, daunorubicin, decitabin, decitabine Doxorubicin, Ephaproxial, Ereschromol, Elsamitorcin, Enocitabine, Epirubicin, Estramstin, Etogluside, Etoposide, Floxuridine, Fludarabin, Fluorouracil (5FU), Fotemustin, Gemcitabin, Gliadel Implant, Hydroxycarbamide, Hydroxyurea, Idal , Iphosphamide, irinotecan, irofluben, ixavepyrone, larotaxel, leucovorin, liposomal doxorubicin, liposomal daunorubicin, ronidamine, romustin, lucanton, mannosulfan, massoprocol, melphalan, mercaptopurine, mesna, methotrexate, aminolexate Mitoguazone, mittan, mitomycin, mitoxanthrone, nedaplatin, nimustin, oblimersen, omacetaxin, altertaxel, oxaliplatin, paclitaxel, pegasuparagase, pemetrexed, pentostatin, pirarubicin, pixantron, plycamycin, porphyramsodium, predoxorubicin , Lanimustin, rubitecan, sapacitabine, semstin, citimagene sera denovec, strataplatin, streptozocin, taraporphin, tegafur-uracil, temoporfin, temozolomide, teniposide, tesetaxel, testlactone, tetranitrate, thiotepa Tipifanib, Topotecan, Travectedin, Triadicon, Triethylenemelamine, Triplatin, Tretinoin, Treosulfan, Trophosphamide, C. Included are ramstin, valrubicin, verteporphyrin, vinblastine, vincristine, vindesine, vinflunin, vinorelbine, vorinostat, sorbicin, and other cell division inhibitors or cytotoxic agents described herein.

Since some drugs work better when combined than when they are used alone, two or more drugs are often administered simultaneously. Frequently, two or more chemotherapeutic agents are used as combination chemotherapeutic agents. In some embodiments, chemotherapeutic agents (including combination chemotherapies) may be used in combination with the compounds described herein.

Targeted therapy Targeted therapy consists of the use of agents that are specific for the disordered proteins in cancer cells. Small molecule targeted therapies are generally inhibitors of mutated, overexpressed, or otherwise important protein enzyme domains in cancer cells. Notable examples are axitinib, bostinib, sedilanib, dasatinib, erlotinib, imatinib, gefitinib, lapatinib, restaulutinib, nilotinib, semaxanib, nilotinib, semaxanib, sorafinib, sunitinib Also included are cyclin-dependent kinase inhibitors such as sericicrib. Monoclonal antibody therapy is another strategy in which the therapeutic agent is an antibody that specifically binds to a cancer cell surface protein. Examples include the anti-HER2 / neu antibody trastuzumab (Herceptin®), which is typically used in breast cancer, and the anti-CD20 antibodies rituximab and tositumomab, which are typically used in a variety of B cell malignancies. Can be mentioned. Other exemplary antibodies include cetuximab, panitumab, trastuzumab, alemtuzumab, bevacizumab, edrecolomab, and gemtuzumab. Illustrative fusion proteins include aflibercept and denylokin diftitox. In some embodiments, targeted therapies may be used in combination with the compounds described herein. For example, Gleevec (Vignarian Wang 2001).

Targeted therapies may also include small peptides as "automated inducers" that can bind to cell surface receptors around the tumor or to the affected extracellular matrix. Radionuclides attached to these peptides (eg, RGD) will eventually kill cancer cells if the nuclide decays near the cell. An example of such a treatment method is BEXXAR®.

Angiogenesis The compounds and methods described herein may be used to treat or prevent angiogenesis-related diseases or disorders. Diseases associated with angiogenesis include cancer, cardiovascular disease, and macular degeneration.

Angiogenesis is a physiological process that involves the growth of new blood vessels from existing blood vessels. Angiogenesis is a normal and essential process in growth and development, as well as wound healing and granulation tissue. But it is also an essential step in the transition of tumors from dormant to malignant. Angiogenesis may be a target for addressing diseases characterized by either poor angiogenesis or an abnormal vasculature.

The application of certain compounds, which may inhibit or induce neovascularization in the body, may also help fight against such diseases. The presence of blood vessels at sites where they should not be present can also affect the mechanical properties of the tissue, increasing the likelihood of damage. The absence of blood vessels during repair or in otherwise metabolically active tissue may interfere with repair or other essential functions. Some diseases, such as ischemic chronic wounds, are the result of damage or inadequate angioplasty and are treated by the local growth of blood vessels, thus supplying new nutrients to the site and promoting repair. You may. Other diseases, such as age-related macular degeneration, may also result from the local growth of blood vessels that interfere with normal physiological processes.

Vascular Endothelial Growth Factor (VEGF) is a major cause of angiogenesis and has been demonstrated to increase the number of capillaries in a given network. Upregulation of VEGF is a major component of the physiological response to exercise, and its role in angiogenesis is believed to be a possible treatment for vascular injury. In in vitro experiments, VEGF is a potent stimulant of angiogenesis because in the presence of this growth factor, the seeded endothelial cells proliferate and migrate, eventually forming a vascular structure similar to capillaries. Clearly demonstrate that.

Tumors induce angiogenesis (angiogenesis) by secreting various growth factors (eg, VEGF). Growth factors such as bFGF and VEGF can induce capillary growth in tumors, and some researchers believe that this provides the necessary nutrients to allow tumor growth.

Angiogenesis corresponds to an excellent therapeutic target for cardiovascular disease treatment. This is a powerful physiological process by which our body responds to a decrease in blood supply to vital organs, the generation of new collateral vessels to overcome ischemic injury. is there.

Overexpression of VEGF causes increased vascular permeability in addition to stimulating angiogenesis. In wet macular degeneration, VEGF causes the growth of capillaries in the retina. Increased angiogenesis also causes edema, causing blood and other retinal fluids to leak into the retina, causing vision loss.

Anti-angiogenic therapies include kinase inhibitors that target vascular endothelial growth factor (VEGF) such as snitinib and sorafenib; or monoclonal antibodies or receptors against VEGF or VEGF receptors, including bevasizumab or VEGF-Trap. "Decoy"; or salidamide or an analog thereof (lenaridomido, pomalidemid); or an agent that targets a non-VEGF angiogenic target such as fibroblast endothelial growth factor (FGF), angiopoetin, or angiostatin or endostatin. Can be mentioned.

Epigenetics The compounds and methods described herein may be used to treat or prevent epigenetics-related diseases or disorders. Epigenetics is the study of hereditary changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence. An example of epigenetic changes in eukaryotes is the process of cell differentiation. During morphogenesis, stem cells become various embryonic cell lines, which in turn become fully differentiated cells. In other words, a single fertilized egg cell changes into a number of cell types, including neurons, muscle cells, epithelium, blood vessels, etc., as it continues to divide. This is done by activating some genes while suppressing other genes.

Epigenetic changes are conserved as cells divide. Most epigenetic changes occur only in the life of one individual, but some epigenetic changes are 1 if mutations in the DNA are caused in the fertilized sperm or egg cell. It is inherited from one generation to the next. Specific epigenetic processes include paramutation, bookmarking, imprinting, gene silencing, X-chromatin inactivation, position effect, reprogramming, transvation, maternal effect, carcinogenesis progression, and many teratogenic factors. Effects of histone modification and regulation of heterochromatin, and technical limitations affecting particulation and cloning.

Illustrative disorders associated with epigenetics include ATR Syndrome, Fragile X Syndrome, ICF Syndrome, Angelman Syndrome, Prader-Willis Syndrome, BWS, Rett Syndrome, α-Saracemia, Cancer, Examples include leukemia, Rubinstein-Tevi syndrome, and Coffin-Raleigh syndrome.

The first human disease associated with epigenetics is cancer. Researchers have found that affected tissue from patients with colorectal cancer has less DNA methylation than normal tissue in the same patient. Since methylated genes are typically switched off, loss of DNA methylation can lead to abnormally high gene activation by altering chromatin placement. On the other hand, overmethylation can undo the action of the protective tumor suppressor gene.

DNA methylation occurs at the CpG site, and in mammals the majority of CpG cytosine is methylated. However, near the promoter region is a series of DNA with higher concentrations of methylation-free CpG sites in normal cells (known as CpG islands). In cancer cells, these CpG islands are overmethylated, thereby switching off genes that should not be decompressed. This abnormality is a characteristic that symbolizes the epigenetic changes that occur in tumors and occurs early in the development of cancer. Overmethylation of CpG islands can cause tumors by switching off tumor suppressor genes. In fact, these types of changes may be more common in human cancers than mutations in DNA sequences.

More epigenetic changes do not alter the DNA sequence, but they can cause mutations. About half of the genes that cause familial or hereditary forms of cancer are switched off by methylation. Most of these genes normally suppress tumorigenesis and repair DNA, including O6-methylguanine-DNA methyltransferase (MGMT), MLH1 cyclin-dependent kinase inhibitor 2B (CDKN2B), and RASSF1A. Help. For example, hypermethylation of the MGMT promoter increases the number of G-to-A mutations.

Hypermethylation can also result in instability of microsatellite, which is a repetitive DNA sequence. Microsatellites are common in normal individuals, and they usually consist of repetitions of dinucleotide CA. Excessive methylation of the promoter of the DNA repair gene MLH1 can destabilize microsatellite and prolong or shorten it. Microsatellite instability has been associated with a number of cancers, including colorectal, endometrium, ovarian, and gastric cancers.

Fragile X syndrome is the most frequently inherited psychiatric disorder, especially in men. Both men and women can develop this condition, but since men have only one X chromosome, one fragile X has a more significant effect on men. In fact, Fragile X Syndrome occurs in about 1 in 4,000 men and 1 in 8,000 women. People with this syndrome have severe intellectual disability, delayed language development, and "autism-like" behavior.

Fragile X Syndrome is named after the appearance of the X chromosome portion containing the genetic abnormality under a microscope. It usually looks like it is hanging with a thread and is easily broken. The syndrome is caused by an abnormality in the FMR1 (fragile X mental retardation 1) gene. People without Fragile X Syndrome have 6-50 repeats of the tribase CGG in the FMR1 gene. However, individuals with more than 200 repeats have complete mutations and usually exhibit symptoms of the syndrome. Too much CGG causes CpG islands to become methylated in the promoter region of the FMR1 gene; under normal conditions, they are not methylated. This methylation switches off the gene and stops the FMR1 gene from producing a key protein called the fragile X mental retardation protein. A decrease in this particular protein causes Fragile X Syndrome. CGG amplification mutations are attracting attention as the cause of Fragile X, but the epigenetic changes associated with FMR1 methylation are the true culprit of the syndrome.

Fragile X syndrome is not the only disorder associated with mental retardation with epigenetic changes. Other such medical conditions include Rubinstein-Taybi, Coffin-Lowry, Prader-Willi, Angelman, Beck with Wiedemann, ATR-X, and Rett Syndrome.

Epigenetic therapies include inhibitors of enzymes that control epigenetic modifications, specifically DNA methyltransferases and histone deacetylases that have promising antitumorogenic effects on some malignancies. Included are antisense oligonucleotides and siRNA.

Immunotherapy In some embodiments, the compounds described herein are administered with immunotherapy. Cancer immunotherapy refers to a diverse set of treatment strategies designed to induce the patient's own immune system to fight the tumor. Modern methods of producing an immune response against tumors include intravesical BCG immunotherapy for superficial bladder cancer, the prostate cancer vaccine Provenge, and the immune response in patients with renal cell carcinoma and melanoma. Included is the use of interferon and other cytokines to induce.

Allogeneic hematopoietic stem cell transplantation can be considered a form of immunotherapy because the donor's immune cells often attack the tumor with a graft-versus-tumor effect. In some embodiments, immunotherapeutic agents may be used in combination with the compounds described herein.

Hormone Therapy In some embodiments, the compounds described herein are administered with hormone therapy. The growth of certain cancers can be suppressed by providing or blocking certain hormones. Common examples of hormone-sensitive tumors include certain types of breast and prostate cancer, as well as certain types of leukemia in response to certain retinoids / retinoic acid. Removing or blocking estrogen or testosterone is often an important additional treatment. For certain cancers, administration of hormonal agonists such as progestogen may be therapeutically beneficial. In some embodiments, hormonal therapeutic agents may be used in combination with the compounds described herein.

Hormone therapy agents include administration of hormone agonists or hormonal antagonists, including retinoid / retinoic acid, compounds that inhibit estrogen or testosterone, and administration of progestogen.

Inflammation and Autoimmune Diseases The compounds and methods described herein may be used to treat or prevent inflammation-related diseases or disorders, especially in humans and other mammals. The compounds described herein may be administered before, during, or after the onset of inflammation. When used prophylactically, the compound is preferably provided prior to any inflammatory response or symptom. Administration of the compound may prevent or reduce the inflammatory response or symptoms. Exemplary inflammatory conditions include, for example, polysclerosis, rheumatoid arthritis, psoriatic arthritis, degenerative joint disease, spondolorearthropathy, other seronegative inflammatory bowel inflammation, rheumatoid polymyopathy, and more. Vasculitis (eg giant cell arthritis, ANCA + vasculitis), gouty arthritis, systemic erythematosus, juvenile arthritis, juvenile rheumatoid arthritis, osteoarthritis, osteoporosis, diabetes (eg insulin-dependent diabetes or juvenile-onset diabetes) , Menstrual spasm, cystic fibrosis, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, mucinous colitis, ulcerative colitis, gastric inflammation, esophagitis, pancreatitis, peritonitis, Alzheimer's disease, shock, tonic spine Inflammation, gastrointestinal inflammation, conjunctivitis, vasculitis (acute or chronic), multi-organ injury syndrome (eg secondary to sepsis or trauma), myocardial infarction, atherosclerosis, stroke, reperfusion injury (eg cardiopulmonary bypass or Includes (caused by renal dialysis), acute glomerulonephritis, thermal injury (ie, sunburn), necrotizing enteritis, granulocyte transfusion-related syndrome, and / or Schegren's syndrome. Exemplary skin inflammatory conditions include, for example, eczema, atopic dermatitis, contact dermatitis, urticaria, scleroderma, psoriasis, and skin diseases with acute inflammatory components.

In another embodiment, the compounds and methods described herein include asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity, emphysema, chronic bronchitis, acute dyspnea syndrome, and any chronic It may be used to treat or prevent allergic and respiratory conditions, including obstructive pulmonary disease (COPD). The compounds may be used to treat chronic hepatitis infections, including hepatitis B and hepatitis C.

In addition, the compounds or methods described herein may be used to treat autoimmune diseases and / or inflammation. Organ tissue autoimmune disease (eg Reynaud syndrome), strong skin disease, severe myasthenia, transplant rejection, endotoxin shock, sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune thyroiditis, grape membrane It has been associated with autoimmune diseases such as inflammation, systemic erythematosus, Addison's disease, multiglandular autoimmune disease (also known as multiglandular autoimmune syndrome), and Graves' disease.

In certain embodiments, the compounds described herein can be used to treat multiple sclerosis. In certain embodiments, the compounds used to treat multiple sclerosis are Compound 1, (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1, 2,4-Triazole-1-yl) -1- (3,3-difluoroazetidine-1-yl) prop-2-ene-1-one).

Combination Therapy In certain embodiments, the compounds described herein may be administered alone or in combination with other compounds useful in treating or preventing inflammation. Exemplary anti-inflammatory drugs include, for example, steroids (eg cortisol, cortisol, fludrocortisone, prednisone, 6 [α] -methylprednisolone, triamcinolone, betamethasone or dexamethasone), non-steroidal anti-inflammatory drugs (NSAIDS). (For example, aspirin, acetaminophen, tolmethin, ibuprofen, mephenamic acid, pyroxicum, nabmeton, lofecoxib, selecoxib, etodrac or nimeslide). In another embodiment, the other therapeutic agent is an antibiotic (eg, vancomycin). , Penicillin, Amoxycillin, Ampicillin, Cefotaxim, Ceftriaxone, Cefixim, Rifampin Metronidazole, Doxycycline or Streptomycin. In another embodiment, the other therapeutic agent is a PDE4 inhibitor (eg, loflumirast or loliplum). In form, the other therapeutic agent is an anti-histamine (eg, cyclidine, hydroxydine, promethazine or diphenhydramine); in another embodiment, the other therapeutic agent is an anti-malaria agent (eg, artemicinin, arteter, artesnate) (Artsunate), chlorokin phosphate, meflokin hydrochloride, doxicycline hydrochloride, proguanyl hydrochloride, atobacone or halophantrin). In one embodiment, the other compound is drotrecogin alpha.

Further examples of anti-inflammatory agents include, for example, acecrofenac, codeine, e-acetamide caproic acid, acetaminophen, acetaminosalol, acetanilide, acetylsalicylic acid, S-adenosylmethionine, alcrophenac, alcromethasone, alfentanyl, Algeston, allylprozine, aluminoprofen, alloxypurine, alphaprodine, bis (acetylsalicylate) aluminum, amcinonide, amphenac, aminochlortenoxazine, 3-amino-4-hydroxybutyric acid, 2-amino-4-picolin, amino Propyrone, aminopyrin, amixetrin, ammonium salicylate, ampyroxycam, amtormethingacyl, anileridine, antipyrine, anthrafenin, apazone, beclomethasone, bendazac, benolylate, benoxaprofene, benzpiperylone, benzidamine, benzylmorphine, velmoprofen , Betamethasone, 17-betamethasone valerate, vegitramide, [α] -bisabolol, bromfenac, p-bromoacetanilide, 5-bromosalicylic acid acetate, bromosaligenin, busetin, bucloxic acid, bucolome, budesonide, bufexamac, bumadizone, buprenorfin, Butoacetin, butibphen, butorphanol, carbamazepine, carbiphen, carprofen, calsalam, chlorobutanol, chloroprednison, chlortenoxazine, choline salicylate, cincophene, symmethacin, ciramador, clidanac, clobetazole, crocortron, clomethacin, chronitazen, chlorixin Noll, clove, codeine, methyl codeine bromide, codeine phosphate, codeine sulfate, cortisone, cortibazole, clopropamide, crotetoamide, cyclazosin, defrazacoat, dehydrotestosterone, desomorphine, desonide, desoxymethasone, dexamethasone, dexamethasone-21-isonicotine Acid, dexoxadrol, dextromoramide, dextropropoxyphene, deoxycorticosterone, dezosin, diampromide, diamorphone diamorphone, diclofenac, diphenamizol, diphenpyramide, diflorazone, diflucortron, diflunisar, diflu Predonato, dihydrocodeine enol acetate, dihydromorph Ne, dihydroxyaluminum acetylsalicylate, dimenoxador, dimeheptanol, dimethylthianbutene, dioxafetyl butyrate, dipipanone, diprocetyl, dipyrone, ditazole, droxycam, emorphazone, enphenamic acid, enoxolone, epilyzole, eptazocin, ethelsalate, etenzamid, etenzamid. Buten, ethylmorphine, etdrac, etofenamate, etnitazen, eugenol, fervinac, fenbufen, fenclosic acid, fendsar, phenoprofen, fentanyl, fentiazac, fepradinol, feprazone, floctaphenin, fluazacoat, furchloronide, flufenamic acid, flumethasone. , Flunicin, Flunoxaprofen, Fluosinoloneacetonide, Fluosinide, Fluosinoloneacetonide, Fluocortinbutyl, Fluocortrone, Fluoleson, Fluorometron, Fluperolone, Flupiltin, Flupredonidene, Fluprednisolone, Fluproquazone, Fullland lenolide, flurubiprofen, fluticason, formocortal, phosphosar, gentidic acid, graphenine, glucamethacin, glycol salicylate, guaiazulene, halcinonide, halobetasol, halomethasone, halopredonone, heroine, hydrocodon, hydrocortamate, hydrocortisone, hydrocortisone Hydrocortisone Acid, Hydrocortisone Hemicohactate, Hydrocortisone-21-Riginate, Hydrocortisone Shipionate, Hydromorphine, Hydropethidine, Evephenac, Ibuprofen, Ibuproxam, Imidazole Salicylate, Indomethacin, Indoprofen, Isophezolac, Isoflupredone, Isoflupredone acetate , Isometadone, isonyxin, isoxepac, isoxycam, ketobemidone, ketoprofen, ketrolac, p-lactophenetide, refetamine, levallorphan, levorphanol, levofenacyl-morphan, lofentanyl, lonazolac, lornoxicum, loxoprofen, acetylsalicylic acid , Mephenamic acid, meroxycam, meperidine, meprednison, meptazinol , Mesalamine, Metazosin, Mesadone, Metotrimeprazine, Methylprednisolone, Methylprednisolone acetate, Methylprednisolone sodium succinate, Methylprednisolone sleptnert, Methiadic acid, Metphorin, Metopon, Mofebutazone, Mofezorak, Momethazone, Morazone, Morhine , Morphine sulfate, morpholine salicylate, mylofin, nabmeton, nalbufin, nalolfin, 1-naphthyl salicylate, naproxene, nalcein, nehopam, nicomorphine, niphenazone, niflumic acid, nimeslide, 5'-nitro-2'-propoxyacetoanilide, norlebolfanol , Normetadon, normorphine, norpipanone, orsalazine, achen, oxaceptol, oxamethasine, oxaprodin, oxycodon, oxymorphone, oxyphenbutazone, papabeletam, parameterzone, paraniline, pulsalmid, pentazocin, perisoxal, phenacetin, phenadoxone, phenazocin, phenazopyridine hydrochloride , Phenocol, phenoperidine, phenopyrazone, phenomorphan, phenylacetylsalicylate, phenylbutazone, phenylsalicylate, pheniramidol, picketprofen, piminodin, pipebzone, piperiron, pyrazolac, pyritranide, pyroxycam, pyruprofen, planoprofen Bate, prednisolone, prednison, prednival, prednylidene, progourmet tacin, proheptazine, promedol, propacetamol, properidine, propyram, propoxyphene, propiphenazone, procazone, protidic acid, proxazole, lamiphenazone, remifentanyl, methylsulfate lymazolium , Salicylamide, salicylamide o-acetic acid, salicylic acid, salicylic acid, salsalate, salberin, simetrid, spentanyl, sulfasalazine, slindac, superoxide dismutase, sprofene, sxibuzone, tarniflumate, tenidap, tenoxycam, terofenamate, Tetlandrin, thiazolinobtazone, thiaprofenic acid, thialamide, tilydin, tynolysine, thixocortor, tolfenamic acid, tolmethin, tramadol, triamsinolone, triamsinolone acetnide, tropesin, biminol, xembucin, ximoprofen, Examples include zaltoprofen and zomepillak.

In one embodiment, the compounds described herein may be administered with a selective COX-2 inhibitor to treat or prevent inflammation. Exemplary selective COX-2 inhibitors include, for example, delacoxib, parecoxib, celecoxib, valdecoxib, rofecoxib, etoricoxib, and lumiracoxib.

In some embodiments, the provided compound is administered in combination with an anthracycline or Topo II inhibitor. In certain embodiments, the provided compound is administered in combination with doxorubicin (Dox). In certain embodiments, the provided compound is administered in combination with bortezomib, more broadly including carfilzomib. It has been surprisingly found that the provision of compounds in combination with Dox or bortezomib results in a synergistic effect (ie, outweighs the additive effect).

Viral Infections The compounds and methods described herein may be used to treat or prevent viral infection-related diseases or disorders, especially in humans and other mammals. The compounds described herein may be administered before, during, or after the outbreak of a viral infection. When used prophylactically, the compound is preferably provided prior to any viral infection or symptoms thereof.

Exemplary viral diseases include acute febrile pharyngitis, herpes labialis, epidemic keratoconjunctivitis, infant gastroenteritis, coxsackie infection, infectious mononuclear disease, Berkit lymphoma, acute hepatitis, chronic hepatitis, liver cirrhosis, liver. Cellular cancer, primary HSV-1 infections (eg gingival stomatitis in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infections (eg herpes labialis) and herpes simplex (cold) soles), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononuclear disease, giant cell inclusion disease, Kaposi sarcoma, multicentric Castleman's disease, primary exudation Fluid lymphoma, AIDS, influenza, Rye syndrome, ulcer, post-infectious encephalomyelitis, mumps cold, hyperplastic epithelial lesions (eg, vulgaris, flattened, sole, and anal genital vulgaris, herpes labialis, herpes labialis) Herpes labialis), cervical cancer, herpes labialis, cloop, pneumonia, herpes labialis, infectious disease, gray myelitis, mad dog disease, influenza-like syndrome, severe herpes labialis with pneumonia, wind rash, congenital herpes, Examples include herpes labialis and herpes labialis.

Exemplary pathogenic viruses include adenovirus, coxsackie virus, dengue virus, encephalitis virus, Epstein bar virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, simple herpesvirus type 2, Cytomegalovirus, human herpesvirus type 8, human immunodeficiency virus, influenza virus, measles virus, mumps cold virus, human papillomavirus, parainfluenza virus, poliovirus, mad dog disease virus, respiratory follicles virus, wind rash virus, varicella herpes zoster Examples include viruses, West Nile virus, Dungee, and yellow fever virus. Pathogenic viruses also include viruses that cause resistant viral infections.

Antiviral drugs are a class of drugs that are specifically used to treat viral infections. Antiviral activity is generally classified into one of three mechanisms. Interference with the ability of the virus to invade target cells (eg amantadine, rimantadine, and preconalyl), suppression of virus synthesis (eg nucleoside analogs such as acyclovir and zidovudine (AZT)), and suppression of virus release (eg zanamivir) And oseltamivir).

Ophthalmology The compounds and methods described herein may be used to treat or prevent ophthalmology disorders. Exemplary macular disorders include macular edema (diabetic and non-diabetic macular edema), age-related moist and dry macular degeneration, age-related disciform macular degeneration, and cysts. Macular edema, eyelid edema, retinal edema, diabetic retinopathy, chorioretinosis, angiogenic macular disease, angiogenic glaucoma, vegetative inflammation, irisitis, retinal angiitis, endophthalmitis, total ophthalmitis, metastatic eye Flame, chorioretinitis, retinal pigment epitheliitis, conjunctivitis, macular inflammation, macular inflammation, epimyelitis, optic neuritis, retrobulbar optic neuritis, keratitis, eyelid inflammation, exudative retinal detachment, macular ulcer, conjunctival ulcer , Chronic monetary keratitis, eye diseases associated with hypoxia or ischemia, premature infant retinopathy, proliferative diabetic retinopathy, polypoid chorioangiopathy, retinal hematoma-like growth, retinal artery occlusion, retinal vein Obstruction, Coates' disease, familial exudative vitreous retina, pulseless disease (high anxiety), Eel's disease, antiphospholipid antibody syndrome, leukemia retina, blood hyperviscosity syndrome, macroglobulinemia, interferon Included are retina, hypertensive retina, radiation retina, macular epithelial stem cell insufficiency, and macula.

Other ophthalmic diseases that can be treated using the compounds and methods described herein include proliferative vitreoretinopathy and chronic retinal withdrawal.

Inflammatory eye diseases can also be treated using the compounds and methods described herein.

Neurodegenerative Diseases Neurodegeneration is a comprehensive term that refers to the progressive loss of nerve cell structure or function, including the death of nerve cells. Many neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and Huntington's disease, occur as a result of the neurodegenerative process. As research progressed, numerous similarities emerged that correlate these diseases at the intracellular level. The discovery of these similarities provides hope for advances in treatments that can improve multiple diseases at the same time. There are many similarities between different neurodegenerative disorders, including atypical protein assembly and induced cell death.

Alzheimer's disease is characterized by the loss of neurons and synapses within the cerebral cortex and certain subcortical areas. This loss results in gross atrophy of the affected area, including degeneration in the temporal and parietal lobes, and in the frontal cortex and parts of the cingulate gyrus.

Huntington's disease causes astrogliosis and loss of medium-sized spiny neurons. Regions of the brain are affected by their structure and the neuronal types they contain, and decrease in size as they lose cells cumulatively. The affected areas are primarily in the striatum, but also in the frontal and temporal cortex. The subthalamic nucleus of the striatum sends control signals to the globus pallidus, which initiates and regulates movement. Thus, a decrease in signal from the subthalamic nucleus causes a decrease in the initiation and regulation of movement, resulting in the movement characteristic of the disease. Exemplary therapeutic agents for Huntington's disease include tetrabenandine, neuroleptics, benzodiazepines, amantadine, remasemide, valproic acid, selective serotonin reuptake inhibitors (SSRIs), mirtazapine, and antipsychotics.

The mechanism by which brain cells are lost in Parkinson's disease may also consist of an abnormal accumulation of the ubiquitin-bound protein α-synuclein in damaged cells. The α-synuclein-ubiquitin complex cannot be induced in the proteasome. This protein accumulation forms proteinaceous cytoplasmic inclusion bodies called Lewy bodies. Recent studies on the etiology of the disease show that α-synuclein-induced death of dopaminergic neurons results in a defect in the mechanism by which proteins are transported between the two major organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. It shows that it is caused. Certain proteins, such as Rab1, may also reverse this defect caused by α-synuclein in animal models. Illustrative Parkinson's disease therapies include levodopa, dopamine agonists such as bromocriptine, pergolide, pramipexol, ropinirole, pyribezil, cabergoline, apomorphine, and rislide, and dopa decarboxylate inhibit. , MAO-B inhibitors such as selegilene and rasagiline, anticholinergic agents, and amantadine.

Amyotrophic lateral sclerosis (ALS / Lou Gehrig's disease) is a disease in which motor neurons are selectively degenerated. Exemplary ALS therapies include riluzole, baclofen, diazepam, trihexyphenidyl, and amitriptyline.

Other exemplary neurodegenerative therapeutic agents include antisense oligonucleotides and stem cells.

Wound healing Wounds are a type of condition characterized by cell or tissue damage. Wound healing is optimally a dynamic process that results in the restoration of tissue integrity and function. The wound healing process consists of three overlapping stages. The first stage is the inflammatory phase characterized by homeostasis, platelet aggregation, and degranulation. Platelets as the first response release multiple growth factors to mobilize immune cells, epithelial cells, and endothelial cells. The inflammatory phase typically occurs over 0-5 days. The second stage of wound healing is the proliferative phase during which macrophages and granulocytes invade the wound. Infiltrative fibroblasts begin to produce collagen. The principle features of this period are epithelialization, angiogenesis, granulation tissue formation, and collagen production. The proliferative phase typically occurs over a period of 3-14 days. The third stage is the reconstruction phase in which matrix formation occurs. Fibroblasts, epithelial cells, and endothelial cells continue to produce collagen and collagenase, as well as matrix metalloproteinase (MMP), for remodeling. Collagen cross-linking occurs, causing the wound to contract. The restructuring period typically occurs from day 7 to 1 year.

The compounds and compositions described herein promote wound healing (eg, promote or accelerate wound closure and / or wound healing, reduce scar fibrosis in and / or surrounding wound tissue, and peri-wound. Or it can be used to inhibit apoptosis of nearby cells). Thus, in certain embodiments, the present invention facilitates wound healing within a subject, comprising the step of administering to the subject a compound (eg, a CRM1 inhibitor), or a pharmaceutically acceptable salt or composition thereof. Provide a way to do it. The method does not need to achieve complete healing or closure of the wound; it is sufficient that the method promotes wound closure to any extent. In this regard, the method can be used alone or as an adjunct to other methods for healing injured tissue.

The compounds and compositions described herein are used to treat wounds during the inflammatory (or early), wound healing and proliferation (or mid), and / or wound healing and remodeling (or late) stages. obtain.

In some embodiments, the subject in need of wound healing is a human or an animal such as a rodent such as a horse, pig, or mouse.

In some embodiments, the compounds and compositions described herein that are useful for wound healing are administered topically or systemically, eg, in close proximity to the wound site.

More specifically, the compounds or compositions described herein are adhesive plasters, filling materials, by coating a wound or coated or treated with a compound or composition described herein. , Sutures and the like can be administered to the wound site (optionally in combination with other agents). Thus, the compounds and compositions described herein can be formulated for topical administration to treat surface wounds. Topical preparations include oral (buccal) delivery preparations, skin delivery preparations in which the compounds or compositions described herein are brought into contact with the skin layer (ie, epidermis, dermis, and / or subcutaneous layer). .. Topical delivery systems may be used to administer topical formulations of the compounds and compositions described herein.

Alternatively, the compounds and compositions described herein are, for example, injections of solutions, injections of sustained release formulations, or biodegradable transplants comprising the compounds or compositions described herein. By introduction of the piece, it can be administered at or near the wound site.

The compounds and compositions described herein can be used to treat acute or chronic wounds. Chronic wounds occur when the normal repair process is interrupted. Chronic wounds can result from acute injury as a result of unrecognized persistent infection or inadequate first-line treatment. But most often, chronic injury is the terminal stage of progressive tissue destruction due to venous, arterial, or metabolic vascular disease, pressure ulcers, radiation damage, or tumors.

Chronic wounds do not heal for a variety of reasons, including improper circulation in diabetic ulcers and marked necrosis in burns and infections. In these chronic wounds, the survival or recovery phase is often the rate-determining step. The cells can no longer survive and therefore the initial recovery phase is prolonged by the unfavorable wound bed environment.

Chronic wounds include chronic ischemic skin lesions; scleroderma ulcers; arterial ulcers; diabetic foot ulcers; pressure ulcers; venous ulcers; non-healing lower limb wounds; ulcers due to inflammatory conditions; and / or long-term wounds. However, it is not limited to this.

In certain embodiments, the compounds and compositions described herein are used for diabetic wound healing in a subject or to accelerate lower limb and foot ulcer healing secondary to diabetes or ischemia. Can be done.

In one embodiment, the wound is a trauma. In another embodiment, the wound is a surgical wound (eg, abdominal or gastrointestinal surgical wound). In a further embodiment, the wound is a burn. In yet another embodiment, the wound is the result of radiation exposure.

The compounds and compositions described herein can also be used for healing diabetic wounds, healing gastrointestinal wounds, or healing adhesions resulting from, for example, surgery.

The compounds and compositions described herein can also be used to heal wounds secondary to another disease. In inflammatory skin diseases such as psoriasis and dermatitis, there are numerous skin lesion events secondary to the disease, caused by deep cracks in the skin or by scratching the skin. The compounds and compositions described herein can be used to heal wounds secondary to these diseases, such as inflammatory skin diseases such as psoriasis and dermatitis.

In a further embodiment, the wound is a trauma. In certain embodiments, the trauma is a chronic wound. In another particular aspect, the wound is a vascular wound. In yet another particular aspect, the trauma is an ulcer.

Examples of wounds include abrasions, lacerations, open pneumothorax (ie open pneumothorax), burn wounds, contusions, gunshot wounds, incisions, open wounds, penetrating wounds, puncture wounds, puncture wounds, skewer wounds, puncture wounds, These include, but are not limited to, surgical wounds, subcutaneous wounds, diabetic lesions, or tangential wounds. Additional examples of wounds that can be treated with the compounds and compositions described herein are caused by burns, chemical burns, acute medical conditions or wounds, chemical burns, radiation burns, and exposure to excessive UV radiation. Burns (eg, sunburn); damage to body tissues such as the perineum as a result of labor and childbirth; injuries received during medical procedures such as perineal incision; cutting, incision, epidermis peeling, etc. These include trauma-induced injuries; injuries from accidents; postoperative injuries, as well as chronic medical conditions such as decubitus, floor rubs, diabetes and poor circulation-related medical conditions, and all types of acne. Further wounds include dermatitis such as impetigo, interstitial rash, folliculitis and eczema, wounds following dental surgery; periodontal disease; wounds following trauma; and tumor-related wounds. Still other examples of wounds include animal bites, arterial disease, insect stings and bites, bone infections, injured skin / muscle transplants, necrosis, skin tears or lacerations, skin aging, slow recovery or non-healing. These include surgical incisions, including surgical wounds, intracerebral hemorrhage, aneurysms, cutaneous asthenia, and postoperative infections.

In a preferred embodiment, the wound is from burns, cuts, open wounds, surgical or post-surgical wounds, diabetic lesions, burns, chemical burns, radiation burns, pressure sores, bed sores, and medical conditions associated with diabetes or poor circulation. Selected from the group of

The disclosure also relates to methods and compositions that reduce scar formation during wound healing in a subject. The compounds and compositions described herein can be administered directly to the wound or to cells in close proximity to the wound in an amount effective to reduce scar formation in and / or around the wound.

Wounds include any injury to any part of the subject's body. According to embodiments, methods are provided for improving, reducing, or reducing scar formation in a subject suffering from burns. According to a preferred embodiment, methods are provided for treating hypertrophic scars, reducing their appearance, or reducing their chances of developing in a subject suffering from an acute or chronic wound or injury.

Other Disorders The compounds and compositions described herein also cause dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, pulmonary fibrosis, liver fibrosis, glomerular nephritis, and other nephropathy. It may also be used to treat abnormal tissue growth and fibrotic disorders, including.

Combination Radiation Therapy The compounds and compositions described herein are useful as radiosensitizers. Thus, the compounds and compositions described herein can be administered in combination with radiation therapy. Radiation therapy is the medical use of high-energy irradiation (eg, x-rays, gamma rays, charged particles) to shrink tumors and kill malignant cells and is commonly used as part of cancer treatment. To. Radiation therapy kills malignant cells by damaging their DNA.

Radiation therapy can be delivered to the patient in several ways. Irradiation can be delivered from an external source, such as an external device in the patient's body, as in external beam radiotherapy. External beam radiation therapy for cancer treatment is a radiation source external to the patient, typically either a radioisotope such as ⁶⁰ Co, ¹³⁷ Cs, or a high-energy astrophysical source such as a linear accelerator. To use. External sources produce a parallel beam towards the patient's tumor site. External-source radiation therapy avoids some of the problems of internal-source radiation therapy, but it, along with neoplastic tissue, causes undesired and necessarily large amounts of non-neoplastic or healthy tissue in the radiation beam path. Irradiate.

The detrimental effect of irradiating healthy tissue is the given radiation dose in the neoplastic tissue by projecting an external radiation beam at the patient at various "gantry" angles while covering the tumor site with the beam. Can be lowered while maintaining. Certain volume elements of healthy tissue along the path of the radiation beam change, reducing the total dose of healthy tissue to each of these elements throughout the treatment.

Irradiation of healthy tissue can also be reduced by closely collimating the radiation beam over the entire tumor cross section perpendicular to the radiation beam axis. There are numerous systems that produce such perimeter collimation, some of which utilize multiple sliding shutters that can piecewise produce a radiation opaque mask of any contour.

For administration of external beam irradiation, the dose can be at least about 1 Gray (Gy) fraction, at least once every other day, relative to the treatment volume. In certain embodiments, irradiation is administered to the therapeutic volume at least once daily in at least about 2 Gray (Gy) fractions. In another particular embodiment, the irradiation is administered to the therapeutic volume at least once daily in at least about 2 Gray (Gy) fractions for 5 consecutive days per week. In another particular embodiment, the irradiation is administered to the treatment volume every other day, three times a week, in a 10 Gy fraction. In another particular embodiment, at least a total of about 20 Gy is administered to the patient in need of it. In another particular embodiment, at least a total of about 30 Gy is administered to the patient in need of it. In another particular embodiment, at least about 40 Gy is administered to the patient in need of it.

Patients typically receive external beam therapy 4 or 5 times a week. The entire treatment process usually lasts 1 to 7 weeks, depending on the type of cancer and the purpose of treatment. For example, a dose of 2 Gy / day can be administered to the patient for 30 days.

Internal radiation therapy is localized radiation therapy, where the radiation source is located at the tumor site or affected area. Internal radiation therapy can be delivered by placing the radiation source inside or next to the area requiring treatment. Internal radiation therapy is also referred to as proximity radiation therapy. Proximity irradiation therapy includes intracavitary therapy and interstitial therapy. In intracavitary therapy, a container containing the radiation source is placed in or near the tumor. The radiation source is placed in the body cavity. In interstitial therapy, only the radiation source is placed in the tumor. These sources of radiation can remain permanently within the patient. The radiation source is typically removed from the patient after a few days. The radiation source is in the container.

There are several ways to administer radiopharmaceuticals. For example, radiopharmaceuticals can be administered by targeted or systemic delivery of targeted radioactive complexes such as radiolabeled antibodies, radiolabeled peptides, and liposome delivery systems. In a particular embodiment of targeted delivery, the radiolabeled drug can be a radiolabeled antibody. For example, Cancer Res., Which is incorporated herein by reference. , 2001; 61: 2008-2014 and Goldenber, D.I. M. J. Nucl. Med. , 2002; 43 (5): 693-713.

In another particular embodiment of targeted delivery, the radiopharmaceutical may be administered in the form of a liposome delivery system such as small monolayer vesicles, large monolayer vesicles, and multimembrane vesicles. Liposomes can be produced from a variety of phospholipids such as cholesterol, stearylamine or phosphatidylcholine. For example, Emfietzoglou D, Kostarellos K, Sgouros G. et al., The contents of which are incorporated herein by reference. Analytical dosimetry study for the use of radionuclide-liposomes conjugates in international radiotherapy. See J Nucl Med 2001; 42: 499-504.

In yet another specific embodiment of targeted delivery, the radiolabeled drug can be a radiolabeled peptide. For example, Wiener RE, Thakur ML. Radiolabeled peptides in the diagnostics and therapy of oncological diseases. See Apple Radiat Iso 2002 Nov; 57 (5): 749-63.

In addition to targeted delivery, radiopharmaceuticals can be delivered to the target site using proximity therapy. Proximity radiation therapy is a technique that places the radiation source as close as possible to the tumor site. Often the source is inserted directly into the tumor. The radiation source can be in the form of wires, seeds or rods. Generally, cesium, iridium or iodine is used.

Systemic radiation therapy is another type of radiation therapy that involves the use of radioactive substances in the blood. Systemic radiation therapy is a form of targeted therapy. In systemic radiation therapy, the patient typically ingests or is injected with a radioactive substance, such as radioactive iodine, or a radioactive substance bound to a monoclonal antibody.

As defined herein, "radioactive drug" refers to a drug containing at least one radioradioactive isotope. Radiopharmaceuticals are customarily used in nuclear medicine for the diagnosis and / or treatment of various diseases. Radiolabeled pharmaceuticals, such as radiolabeled antibodies, contain radioisotopes (RIs) that act as radiation sources. As intended herein, the term "radioactive isotope" includes metallic and non-metal radioisotopes. Radioisotopes are selected based on the medical use of radiolabeled pharmaceuticals. When the radioisotope is a metal radioisotope, a chelating agent is typically used to bind the metal radioisotope to the molecular residue. When the radioisotope is a non-metal radioisotope, the non-metal radioisotope typically binds directly to the molecular residue or by a linker.

As used herein, a "metal radioisotope" is any suitable metal radioisotope useful in in vivo or in vitro treatment or diagnostic procedures. Suitable metal radioisotopes include Actinium-225, Antimon-124, Antimon-125, Arsenic-74, Thallium-103, Thallium-140, Berylium-7, Bismuth-206, Bismuth-207, Bismuth-212, Bismuth. -213, Cadmium-109, Cadmium-115m, Calcium-45, Cerium-139, Cerium-141, Cerium-144, Cesium-137, Chromium-51, Cobalt-55, Cobalt-56, Cobalt-57, Cobalt-58 , Cobalt-60, Cobalt-64, Copper-60, Copper-62, Copper-64, Copper-67, Elbium-169, Europium-152, Gallium-64, Gallium-67, Gallium-68, Gadrinium-153, Gadrinium -157 Gold-195, Gold-199, Hafnium-175, Hafnium-175-181, Holmium-166, Thallium-110, Indium-111, Iridium-192, Iron 55, Iron-59, Crypton-85, Lead-203 , Lead-210, Lutetium-177, Manganese-54, Mercury-197, Mercury-203, Molybdenum-99, Neodim-147, Neptunium-237, Nickel-63, Niob-95, Bismuth-185 + 191, Palladium-103, Palladium -109, Platinum-195m, Placeozim-143, Promethium-147, Promethium-149, Protoactinium-233, Radium-226, Renium-186, Renium-188, Rubidium-86, Luthenium-97, Luthenium-103, Luthenium- 105, ruthenium-106, samarium-153, scandium-44, scandium-46, scandium-47, selenium-75, silver-110m, silver-111, sodium-22, strontium-85, strontium-89, strontium-90, Io-35, Tantal-182, Technotium-99m, Tellur-125, Tellur-132, Thallium-204, Thallium-228, Thallium-232, Thallium-170, Tin-113, Tin-114, Tin-117m, Titanium- 44, Tungsten-185, Vanadium-48, Vanadium-49, Itterbium-169, Ittrium-86, Ittrium-88, Ittrium-90, Ittrium-91, Zinc-65, Zircon-89, and Zircon-95. , Limited to this It is not something that will be done.

As used herein, a "non-metal radioisotope" is any suitable non-metal radioisotope (non-metal radioisotope) useful in in vivo or in vitro therapeutic or diagnostic procedures. Suitable non-metal radioisotopes include iodine-131, iodine-125, iodine-123, phosphorus-32, astatine-21, fluorine-18, carbon-11, oxygen-15, bromine-76, and nitrogen-13. However, it is not limited to this.

A variety of factors need to be considered to identify the most suitable isotopes for radiation therapy. These include tumor uptake and retention, blood clearance, irradiation delivery rate, radioisotope half-life and specific activity, and the feasibility of economical large-scale production of radioisotopes. The point of therapeutic radiopharmaceuticals is to deliver the required dose to tumor cells to achieve cytotoxic or tumor-killing effects without causing unmanageable side effects.

The physical half-life of the therapeutic radioisotope is preferably similar to the biological half-life of the radiopharmaceutical at the tumor site. For example, if the half-life of the radioisotope is too short, most of the decay occurs before the radiopharmaceutical reaches its maximum target / background ratio. On the other hand, a half-life that is too long can cause unwanted radiation doses to normal tissue. Ideally, the radioisotope should have a sufficiently long half-life, achieve a minimum dose rate, and irradiate all cells at the most sensitive stage of the cell cycle. In addition to this, the half-life of the radioisotope must be long enough to give sufficient time for production, release, and transport.

Another practical consideration in selecting radioisotopes for a particular application in tumor treatment is availability and quality. The purity must be sufficiently high and reproducible, as trace amounts of impurities can affect the radiolabeling and radiochemical purity of radiopharmaceuticals.

Target receptor sites in tumors are typically limited in number. Therefore, the radioisotope preferably has a high specific activity. The specific activity mainly depends on the production method. Trace metal contaminants often compete with radioisotopes for chelating agents, and their metal complexes must be minimized to compete with radiolabeled chelating agents for receptor binding. It doesn't become.

Irradiation types suitable for use in the methods of the invention can vary. For example, the irradiation can be electromagnetic or particulate in nature. Electromagnetic radiation useful in the practice of the present invention includes, but is not limited to, X-rays and gamma rays. Microparticle irradiation useful in the practice of the present invention includes, but is not limited to, electron beams (beta particles), proton beams, neutron beams, alpha particles, and negative pions. .. Irradiation can be delivered using conventional radiological treatment devices and methods, and by intraoperative and stereotactic fixation methods. Additional considerations regarding radiation therapy suitable for use in the practice of the present invention are described in Stephen A. et al. Leibel et al. , Textbook of Radiation Oncology (1998) (published by WB Sanders Company), especially in Chapters 13 and 14. Irradiation can also be delivered by other methods, such as targeted delivery, for example by radioactive "seed" or by systemic delivery of the targeted radioactive complex. J. Padawer et al. , Combined Treatment with Radiosestradiol lucantone in Mouse C3HBA Mammary Adenocarcinoma and with Estradiol lucantone in estrogen. J. Radiat. Oncol. Biol. Phys. 7: 347-357 (1981). Other irradiation delivery methods may also be used in practicing the present invention.

Both α and β particle emitters have been investigated for tumor therapy. Alpha particles are particularly good cytotoxic agents because they dissipate large amounts of energy within one or two cell diameters. Beta particle emitters have a relatively long permeation range (2-12 mm in tissue), depending on the energy level. Long permeation ranges are particularly important in solid tumors with heterogeneous blood flow and / or receptor expression. Beta particle emitters result in a more homogeneous dose distribution, even if they are non-uniformly distributed within the target tissue.

In certain embodiments, therapeutically effective amounts of the compounds and compositions described herein are administered in combination with therapeutically effective amounts of radiation therapy to treat cancer (eg, lung cancer such as non-small cell lung cancer). .. The required dose can be determined by one of ordinary skill in the art based on known doses for a particular type of cancer. For example, Cancer Medicine 5th ed. , Edited by R.M. C. Bast et al. , July 2000, BC Decker.

The above disclosure outlines the present invention. A more complete understanding can be obtained with reference to the specific examples below. These examples are described for purposes of illustration only and are not intended to limit the scope of the invention. Depending on the circumstances or, if convenient, morphological changes and equivalent replacements are considered. Although specific terms are used herein, such terms are intended to be descriptive ideas and are not intended to be restrictive.

Abbreviation aq. Aqueous Boc tert-Butoxycarbonyl CH ₂ Cl ₂ dichloromethane DABCO 1,4-diazabicyclo [2.2.2] Octane DIPEA N, N-diisopropylethylamine DMF N, N-dimethylformamide DMSO Dimethyl sulfoxide eq. Equivalent EtOAc Ethyl Acetate EtOH Ethanol h Time HPLC High Performance Liquid Chromatography LCMS Liquid Chromatography Mass Spectrometry LiOH Lithium Hydroxide NMR Nuclear Magnetic Resonance RT Room Room or Retention Time T3P propylphosphonic acid anhydride TFA trifluoroacetate THF tetrahydrofuran

Throughout the following description of such methods, appropriate protecting groups are added to the various reactants and intermediates, where appropriate, in a manner readily understood by those skilled in the art of organic synthesis, followed by then. It is understood that it will be removed. Conventional procedures for using such protecting groups, as well as examples of suitable protecting groups, are described, for example, in "Protective Groups in Organic Synthesis", T.I. W. Green, P. et al. G. M. Wuts, Wiley-Interscience, New York, (1999). It is also understood that the conversion of a group or substituent to another group or substituent by chemical treatment can be performed on any intermediate or final product on the synthetic pathway towards the final product. In, possible types of conversions are limited only by the inherent incompatibility of the other functional groups of the molecule with respect to the conditions or reagents used for the conversion. Those skilled in the art of organic synthesis will readily understand such inherent incompatibilities and how to avoid them by performing the appropriate conversion and synthesis steps in the appropriate order. Examples of transformations are described below, and it is understood that the transformations described are not limited to the general or substituents for which the transformation is exemplified. References and explanations for other suitable transformations can be found in "Comprehensive Organic Transitions-A Guide to Functional Group Preparations" R. et al. C. Larock, VHC Publicers, Inc. (1989). References and explanations for other suitable reactions are described, for example, in organic chemistry textbooks such as "Advanced Organic Chemistry", March, 4th Edition McGraw Hill (1992), or "Organic Synthesis", Smith, McGraw Hill, (1994). Described in. Techniques for purifying intermediates and final products include, for example, normal and reverse phase chromatography on columns or rotating plates, recrystallization, distillation, and liquid-liquid or solid-liquid extraction, which are skilled in the art. Will be easily understood. Substituents and definitions of groups are as in Formula I, unless defined differently. Unless otherwise noted, the terms "room temperature" and "ambient temperature" shall mean temperatures between 16 and 25 ° C. Unless otherwise noted, the term "reflux" shall mean a temperature above the boiling point of the nominated solvent with respect to the solvent used.

Example 1. (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-pivaloylacrylohydrazide (Compound 1) Synthesis of Compound 1 Compound 1 was synthesized according to the following scheme.
3,5-bis (trifluoromethyl) benzothioamide (step 1)
A 2 L three-necked round-bottom flask was loaded with a solution of 3,5-bis (trifluoromethyl) benzonitrile (200 g) in DMF (1 L). The solution was then treated with NaSH (123.7 g, 2.0 eq.) And MgCl ₂ (186.7 g, 1.0 eq.) And the reaction mixture was stirred at room temperature for 3 hours. The mixture was poured into an ice-water slurry (10 L) and the compound was extracted with EtOAc (3 x 1 L). The combined organic layers were washed with aqueous saturated sodium chloride solution (3 x 100 mL), dried over anhydrous Na ₂ SO ₄ , filtered, concentrated under reduced pressure and 205 g of the desired crude 3,5-bis. (Trifluoromethyl) benzothioamide (yield: 90%) was obtained and used in the next step without further purification.

3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole (step 2)
A 5 L three-necked round-bottom flask was loaded with a solution of 3,5-bis (trifluoromethyl) benzothioamide (205.65 g) in DMF (1.03 L). Hydrazine hydrate (73.2 mL, 2.0 eq.) Was added dropwise and the reaction mixture was stirred at room temperature for 1 hour. HCOOH (1.03 L) was added dropwise and the reaction mixture was refluxed at 90 ° C. for 3 hours. After allowing to cool to room temperature, the reaction mixture was poured into saturated aqueous sodium hydrogen carbonate solution (7 L) and extracted with EtOAc (3 x 1 L). The combined organic layers were washed with aqueous saturated sodium chloride solution (3 x 500 mL), dried over anhydrous Na ₂ SO ₄ , filtered, concentrated under reduced pressure (35 ° C., 20 mmHg) and 180 g of crude product. Brought. The crude is stirred with petroleum ether (3 x 500 mL), filtered, dried and obtained as a pale yellow solid, 160 g of the desired 3- (3,5-bis (trifluoromethyl) phenyl) -1H. -1,2,4-triazole was obtained (yield: 75%).

(Z) -Isopropyl3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylate (step 3)
A 2 L three-necked round-bottom flask was loaded with a solution of 3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole (160 g) in DMF (960 mL). The solution was treated with DABCO (127.74 g, 2 eq.), Stirred for 30 minutes, and then isopropyl (Z) -3-iodoacrylate (150.32 g, 1.1 eq.) Was added dropwise. After 1 hour, the reaction mixture was poured into an ice-water slurry (5 L) and extracted with EtOAc (3 x 1 L). The combined organic layers were washed with aqueous saturated sodium chloride solution (3 x 100 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure (35 ° C., 20 mmHg) to produce 250 g crude. The product was obtained and purified by column chromatography (60/120 silica gel) using an ethyl acetate / n-hexane gradient (the column was packed in hexane and the desired compound was 2% EtOAc / n- It started to elute from hexane). Combine the fractions containing the desired compound to make (Z) -isopropyl3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl). An acrylate (138 g, yield: 61%) was obtained.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid (step 4)
(Z) -Isopropyl3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylate in a 5 L three-necked round bottom flask (130 g, 1.0 eq.) Was dissolved in THF (1.3 L). A solution of LiOH (69.3 g, 5.0 eq.) In water (1.3 L) was added dropwise to the solution, the reaction mixture was stirred at room temperature for 4 hours, and then quenched with 400 mL ice-water slurry. , Diluted with aqueous HCl to make it acidic (pH = 2-3). The mixture was extracted with EtOAc (3 x 1 L) and the combined organic layers were washed with aqueous saturated sodium chloride solution, dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to 110 g (Z) -3. -(3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid (yield: 94%), (cis content by LCMS = 90. 0%, trans content = 8.2%) was obtained.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-pivaloylacrylohydrazide (Compound 1)
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in a 50 mL three-necked round-bottom flask (0.2 g, 1.0 eq.) Was dissolved in EtOAc (20 mL), cooled to −60 ° C., and pivalohydrazide (0.08 g, 1.2 eq.) Was added dropwise. T3P (50% in EtOAc) (0.4 mL, 4 eq.) Was added dropwise, followed by DIPEA (0.4 mL, 4 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound, (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl)- N'-pivaloylacrylohydrazide (0.11 g, yield: 43%) was obtained.

Example 2. (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2-morpholinoacetyl) acrylohydrazide Synthesis of (Compound 2) Methyl 2-morpholinoacetic acid (0.25 g, 1.0 eq.) Was dissolved in ethanol (5 mL) at room temperature in a 25 mL three-necked round-bottom flask of 2-morpholinoacethydrazide. Hydrazine hydrate (0.087 g, 1.1 eq.) Was added dropwise at room temperature and the reaction mixture was refluxed at 95 ° C. for 20 hours. The reaction mixture was concentrated under reduced pressure (40 ° C., 20 mmHg) to give crude 2-morpholinoacetohydrazide (0.23 g), which was used in the next step without further purification.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2-morpholinoacetyl) acrylohydrazide (Compound 2)
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in a 50 mL three-necked round-bottom flask (Example 1, Step 4; 0.5 g, 1.0 eq.) Was dissolved in CH ₂ Cl ₂ : EtOAc (20 mL, 2: 1), cooled to -60 ° C., and 2-morpholinoacethydrazide (0). .23 g, 1.0 eq.) Was added dropwise. T3P (50% in EtOAc) (1.27 mL, 1.5 eq.) Was added dropwise, followed by DIPEA (0.96 mL, 2 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound, (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl)- N'-(2-morpholinoacetyl) acrylohydrazide (0.1 g, yield: 14%) was obtained.

Example 3. (Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) -5-methyl-1H- Synthesis of Pyrazole-4-Carbohydrazide (Compound 3) 5-Methyl-1H-Pyrazole-4-Carbohydrazide In a 25 mL sealed tube, ethyl 5-methyl-1H-pyrazole-4-carboxylate (0.25 g, 1. 0eq.) Was dissolved in ethanol (5 mL) at room temperature. Hydrazine hydrate (1 mL, 5 eq.) Was added dropwise at room temperature and the reaction mixture was heated at 120 ° C. for 20 hours. The reaction mixture was concentrated under reduced pressure (40 ° C., 20 mmHg) to give crude 5-methyl-1H-pyrazole-4-carbhydrazide (0.24 g), which was used in the next step without further purification. ..

(Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) -5-methyl-1H- Pyrazole-4-carbohydrazide (Compound 3)
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in a 50 mL three-necked round-bottom flask (Example 1, Step 4; 0.5 g, 1.0 eq.) Was dissolved in EtOAc: EtOH (15 mL, 2: 1), cooled to -60 ° C., 5-methyl-1H-pyrazol-4-. Carbohydrazide (0.24 g, 1.0 eq.) Was added dropwise. T3P (50% in EtOAc) (1.69 mL, 1.5 eq.) Was added dropwise, followed by DIPEA (2 mL, 8 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound and combine (Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1). -Il) acryloyl) -5-methyl-1H-pyrazole-4-carbhydrazide (0.2 g, yield: 42%) was obtained.

Example 4. (Z) -2- (3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) -N-cyclopropylhydrazine carbothioamide Synthesis of (Compound 4) In a 50 mL three-necked round-bottomed flask, (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole- 1-Il) acrylic acid (Example 1, Step 4; 0.5 g, 1.0 eq.) Was dissolved in EtOAc: EtOH (15 mL, 2: 1), cooled to -60 ° C and N-cyclopropyl. Hydrazine carbothioamide (0.22 g, 1.2 eq.) Was added dropwise. T3P (50% in EtOAc) (1.69 mL, 2 eq.) Was added dropwise, followed by DIPEA (1 mL, 4 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound and combine (Z) -2- (3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1- Ill) acryloyl) -N-cyclopropylhydrazine carbothioamide (0.06 g, yield: 9%) was obtained.

Example 5. (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-methyl-N'-(2-morpholino) Synthesis of acetyl) acrylohydrazide (Compound 5) Methyl2-morpholinoacetic acid (0.5 g, 1.0 eq.) Is dissolved in ethanol (5 mL) at room temperature in a 25 mL sealed tube of N-methyl-2-morpholinoacetohydrazide. did. Methylhydrazine (0.16 g, 1.1 eq.) Was added dropwise at room temperature and the reaction mixture was refluxed at 95 ° C. for 48 hours. The reaction mixture was concentrated under reduced pressure (40 ° C., 20 mmHg) to give crude N-methyl-2-morpholinoacetohydrazide (0.27 g), which was used in the next step without further purification.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-methyl-N'-(2-morpholino) Acetyl) Acrylohydrazide (Compound 5)
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in a 50 mL three-necked round-bottom flask (Example 1, Step 4; 0.3 g, 1.0 eq.) Was dissolved in TTHF: EtOAc (15 mL, 2: 1), cooled to -60 ° C., and N-methyl-2-morpholinoacetohydrazide (. 0.23 g, 1.5 eq.) Was added dropwise. T3P (50% in EtOAc) (1.27 mL, 2.5 eq.) Was added dropwise, followed by DIPEA (0.45 mL, 3 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound, (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl)- N'-methyl-N'-(2-morpholinoacetyl) acrylohydrazide (0.052 g, yield: 12%) was obtained.

Example 6. (Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) piperidine-3-carbohydrazide ( Synthesis of Compound 6) Ethylmethylpiperidine-3-carboxylate (1 g, 1.0 eq.) Was dissolved in ethanol (5 mL) at room temperature in a 30 mL sealed tube of piperidine-3-carbohydrazide. Hydrazine hydrate (1.05 g, 3 eq.) Was added dropwise at room temperature and the reaction mixture was heated at 120 ° C. for 20 hours. The reaction mixture was concentrated under reduced pressure (40 ° C., 20 mmHg) to give crude piperidine-3-carbhydrazide (0.8 g), which was used in the next step without further purification.

(Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) piperidine-3-carbohydrazide ( Compound 6)
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in a 50 mL three-necked round-bottom flask (Example 1, Step 4; 0.25 g, 1.0 eq.) Was dissolved in THF: EtOH (15 mL, 2: 1), cooled to -60 ° C., and piperidine-3-carbohydrazide (0.113 g). , 1.1 eq.) Was added dropwise. T3P (50% in EtOAc) (1.69 mL, 4 eq.) Was added dropwise, followed by DIPEA (0.25 mL, 2 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound and combine (Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1). -Il) acryloyl) piperidine-3-carbohydrazide (0.01 g, yield: 2.4%) was obtained.

Example 7. (S, Z) -2-amino-N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl)- Synthesis of 3-methylbutanehydrazide 2,2,2-trifluoroacetic acid (Compound 7) Compound 7 was synthesized by the following scheme.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylohydrazide (step 1)
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in a 50 mL three-necked round-bottom flask (Example 1, step 4; 0.5 g, 1.0 eq.) Was dissolved in THF (10 mL), cooled to −10 ° C., and NMP (0.3 g, 2.1 eq.) Was added. The reaction mixture was stirred for 5 minutes. Isobutyl chloroformate (0.465 g, 2.4 eq.) Was then added and the reaction mixture was stirred for 1 hour. The solid matter formed was filtered and removed. The filtrate was cooled to 0 ° C. and tert-butoxycarbonylhydrazide (0.21 g, 1.1 eq.) Was added. The reaction mixture was allowed to warm to room temperature and stirred for 1 hour. The reaction mixture was poured into an ice-water slurry and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with aqueous saturated sodium chloride solution (25 mL), dried over anhydrous Na ₂ SO ₄ , filtered, concentrated under reduced pressure (25 ° C., 20 mmHg) and 0.5 g of crude product. Brought. The crude product was then dissolved in THF (10 mL), TFA (2 mL) was added dropwise at room temperature and the reaction mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) and the solids formed were ground with pentane to (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl)). -1H-1,2,4-triazole-1-yl) acrylohydrazide (0.25 g, yield: 48.5%) was obtained.

(S) -2-((tert-Butyloxycarbonyl) amino) -3-methylbutanoic acid In a 25 mL three-necked round-bottom flask, (S) -2-amino-3-methylbutanoic acid (0.8 g, 1. 0eq.) Was dissolved in water (4 mL). Sodium hydrogen carbonate (0.63 g, 1.1 eq.) Followed by di-tert-butyl dicarbonate (2.97 g, 2.0 eq.) Was added and the mixture was stirred at room temperature for 2 hours. The reaction mixture was extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with aqueous saturated sodium chloride solution (25 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure (25 ° C., 20 mmHg) to produce 1.2 g crude. The product was obtained and purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (the column was packed in dichloromethane and the desired compound began to elute from 3% methanol / dichloromethane. T). Fractions containing the desired compound were combined to give (S) -2-((tert-butoxycarbonyl) amino) -3-methylbutanoic acid (0.7 g, yield: 47.3%).

(S, Z) -2-amino-N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl)- 3-Methylbutane Hydrazide 2,2,2-Trifluoroacetic Acid (Compound 7)
In a 10 mL round bottom flask, (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylohydrazide (0. 25 g, 1.0 eq.) Is dissolved in THF (5 mL), cooled to -60 ° C., and (S) -2-((tert-butoxycarbonyl) amino) -3-methylbutanoic acid (0.19 g, 1). .3eq.) Was added dropwise. T3P (50% in EtOAc) (0.81 mL, 2 eq.) Was added dropwise, followed by DIPEA (0.48 mL, 4 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound and combine (S, Z) -tert-butyl (1- (2- (3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1). , 2,4-Triazole-1-yl) acryloyl) hydrazinyl) -3-methyl-1-oxobutan-2-yl) carbamate was obtained (0.07 g, yield: 18%). Next, in a 10 mL round bottom flask, (S, Z) -tert-butyl (1- (2- (3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2) , 4-Triazole-1-yl) acryloyl) hydrazinyl) -3-methyl-1-oxobutan-2-yl) carbamate was dissolved in dichloromethane (2 mL). TFA (0.05 mL) was added and the reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give a crude product (0.01 g), which was ground with petroleum ether and dried under reduced pressure to (S, Z) -2-. Amino-N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) -3-methylbutanehydrazide 2,2 , 2-Trifluoroacetic acid (0.006 g, yield: 2%) was obtained.

Example 8. (Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) pyrazine-2-carbohydrazide ( Synthesis of Compound 8) In a 25 mL three-necked round-bottom flask, (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1 -Il) Acrylic acid (Example 1, Step 4; 0.5 g, 1.0 eq.) Is dissolved in dichloromethane (5 mL), cooled to -60 ° C, and pyrazine-2-carbhydrazide (0.216 g, 0.216 g,). 1.1 eq.) Was added. T3P (50% in EtOAc) (3.39 mL, 4 eq.) Was added dropwise, followed by DIPEA (0.5 mL, 2 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound and combine (Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1). -Il) acryloyl) pyrazine-2-carbhydrazide (0.13 g, yield: 19.4%) was obtained.

Example 9. (Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) -1-methylpiperidin-4 -Synthesis of Carbohydrazide (Compound 9) 1-Methylpiperidin-4-Carbohydrazide. Methyl1-methylpiperidine-4-carboxylate (0.2 g, 1.0 eq.) Was dissolved in ethanol (5 mL) at room temperature in a 25 mL sealed tube. Hydrazine hydrate (0.127 g, 2 eq.) Was added dropwise at room temperature and the reaction mixture was heated at 120 ° C. for 20 hours. The reaction mixture was concentrated under reduced pressure (40 ° C., 20 mmHg) to give crude 1-methylpiperidine-4-carbhydrazide (0.145 g), which was used in the next step without further purification.

(Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) -1-methylpiperidin-4 -Carbohydrazide (Compound 9)
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in a 50 mL three-necked round-bottom flask (0.25 g, 1.0 eq.) Was dissolved in EtOAc: THF (15 mL; 2: 1), cooled to -60 ° C. and 1-methylpiperidin-4-carbhydrazide (0.123 g, 1.1 eq.). ) Was added dropwise. T3P (50% in EtOAc) (0.85 mL, 2 eq.) Was added dropwise, followed by DIPEA (0.31 mL, 2.5 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (35 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combined fractions containing the desired compound, (Z) -N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1) -Il) Acryloyl) -1-methylpiperidin-4-carbhydrazide (0.016 g, yield: 4.5%) was obtained.

Example 10. (S, Z) -2-amino-N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl)- Synthesis of 3-methylbutanehydrazide 2,2,2-trifluoroacetic acid (Compound 10) (R) -2-((tert-butoxycarbonyl) amino) -3-methylbutanoic acid. In a 25 mL three-necked round-bottom flask, (R) -2-amino-3-methylbutanoic acid (0.8 g, 1.0 eq.) Was dissolved in water (4 mL). Sodium hydrogen carbonate (0.394 g, 1.1 eq.), Followed by di-tert-butyl dicarbonate (1.86 g, 2.0 eq.) Was added and the reaction mixture was stirred at 2 room temperature for hours. The reaction mixture was extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with aqueous saturated sodium chloride solution (25 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure (25 ° C., 20 mmHg) to 0.75 g of crude product. Obtained and purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (the column was packed in dichloromethane and the desired compound began to elute from 3% methanol / dichloromethane. ). Fractions containing the desired compound were combined to give (R) -2-((tert-butoxycarbonyl) amino) -3-methylbutanoic acid (0.44 g, yield: 47.3%).

(R, Z) -2-amino-N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl)- 3-Methylbutane Hydrazide 2,2,2-Trifluoroacetic Acid (Compound 10)
In a 10 mL round bottom flask, (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylohydrazide (0. 05 g, 1.0 eq.) Is dissolved in THF (5 mL), cooled to -60 ° C., and (R) -2-((tert-butoxycarbonyl) amino) -3-methylbutanoic acid (0.038 g, 1). .3eq.) Was added dropwise. T3P (50% in EtOAc) (0.16 mL, 2 eq.) Was added dropwise, followed by DIPEA (0.095 mL, 4 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound and combine (R, Z) -tert-butyl (1- (2- (3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1). , 2,4-Triazole-1-yl) acryloyl) hydrazinyl) -3-methyl-1-oxobutan-2-yl) carbamate was obtained (0.017 g, yield: 26%). Next, in a 10 mL round bottom flask, (R, Z) -tert-butyl (1- (2- (3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2) , 4-Triazole-1-yl) acryloyl) hydrazinyl) -3-methyl-1-oxobutan-2-yl) carbamate was dissolved in dichloromethane (2 mL). TFA (0.2 mL) was added and the reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give a crude product (0.02 g), which was ground with petroleum ether and dried under reduced pressure to (R, Z) -2-. Amino-N'-(3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acryloyl) -3-methylbutanehydrazide 2,2 , 2-Trifluoroacetic acid (0.007 g, yield: 35%) was obtained.

Example 11. (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (pyrazine-2-yl) ) Synthesis of acetyl) acrylohydrazide (Compound 11) 2- (Pyrazine-2-yl) acetohydrazide In a 25 mL sealed tube, methyl 2- (pyrazine-2-yl) acetate (0.25 g, 1.0 eq. ) Was dissolved in ethanol (5 mL) at room temperature. Hydrazine hydrate (0.33 g, 4 eq.) Was added dropwise at room temperature and the reaction mixture was heated at 120 ° C. for 20 hours. The reaction mixture was concentrated under reduced pressure (40 ° C., 20 mmHg) to give crude 2- (pyrazine-2-yl) acetohydrazide (0.2 g), which was used in the next step without further purification.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (pyrazine-2-yl) ) Acetyl) Acrylohydrazide (Compound 11)
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in a 50 mL three-necked round-bottom flask (0.3 g, 1.0 eq.) Was dissolved in EtOAc: THF (15 mL; 2: 1), cooled to -60 ° C., 2- (pyrazin-2-yl) acetohydrazide (0.129 g, 1). .1eq.) Was added dropwise. T3P (50% in EtOAc) (1.01 mL, 2 eq.) Was added dropwise, followed by DIPEA (0.35 mL, 2.5 eq.), The reaction mixture was stirred at −60 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure (25 ° C., 20 mmHg) to give the crude product, which was purified by column chromatography (60/120 silica gel) using a methanol / dichloromethane gradient (column in dichloromethane). The desired compound began to elute from 3% methanol / dichloromethane). Combine the fractions containing the desired compound, (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl)- N'-(2- (pyrazine-2-yl) acetyl) acrylohydrazide (0.025 g, yield: 5%) was obtained.

Example 12. (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2-morpholino-2-oxoacetyl) ) Synthesis of acetylohydrazide (Compound 12)
Synthesis of ethyl 2-morpholino-2-oxoacetic acid:
Ethyl 2-chloro-2-oxoacetic acid (1.25 g, 9.18 mmol) solution in diethyl ether (5 mL), morpholine (1.0 g, 11.48 mmol) solution in diethyl ether (20 mL), and triethylamine (1). It was added dropwise to .16 g (11.48 mmol) at 0 ° C. The reaction mixture was allowed to reach room temperature and stirred for 2 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The yellow oil was transferred to 25 mL of ice water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 1 g of crude product, which was further used without any purification. Crude yield 47%. ^{1 1} H NMR (400 MHz, CDCl ₃ ) δ 4.33-4.38 (q, 2H), 3.72-3.76 (m, 4H), 3.65-3.68 (m, 2H), 3.47-3.50 (m, 2H), 1.37-1.40 (t, 3H). LCMS m / z 187.93 [M + H] ⁺ , t _R = 0.525 min.

Synthesis of 2-morpholino-2-oxoacetohydrazide:
Ethyl2-morpholino-2-oxoacetic acid (1.0 g, 5.34 mmol) was dissolved in ethanol (7 mL), and hydrazine hydrate (0.267 g, 5.34 mmol) was added dropwise at 0 ° C. The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure to give 0.9 g of crude product, which was used in the next step without further purification. Crude yield 90%. ^{1 1} H NMR (400 MHz, CDCl ₃ ) δ 9.79 (s, 1H), 4.43-4.48 (m, 2H), 3.56-3.61 (m, 4H), 3.40- 3.48 (m, 4H). LCMS m / z 174.16 [M + H] ⁺ , t _R = 2.031 min.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2-morpholino-2-oxoacetyl) ) Synthesis of Acrylohydrazide:
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in THF (3 mL) (0.2 g, A solution of 0.569 mmol) and 2-morpholino-2-oxoacetohydrazide (0.02 g, 0.175 mmol) was cooled to −60 ° C. T ₃ P (0.098 g, 0.569 mmol) (0.50 mL) was added dropwise, followed by DIPEA (0.11 g, 0.854 mmol), followed by stirring at −60 ° C. for 1 hour. The reaction mixture was transferred to 25 mL of ice water and extracted with ethyl acetate (2 x 25 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 0.3 g of crude product, which was chromatographed (0-4% MeOH / CH ₂ Cl). Purified by ₂ ), 0.15 g of (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N' -(2-Molholino-2-oxoacetyl) acrylohydrazide (yield 50%) was obtained. ^{1 1} H NMR (400 MHz, DMSO-d ₆ ) δ 10.70-10.88 (m, 2H), 9.56 (s, 1H), 8.57 (s, 2H), 8.29 (s, 1H), 7.52-7.55 (d, J = 10.4 Hz, 1H), 6.0-6.03 (d, J = 10.4 Hz, 1H), 3.51-3.64 (M, 8H). LCMS m / z 507.25 [M + H] ⁺ , t _R = 2.012 min.

Example 13. (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (3,5-dimethyl) Synthesis of morpholino) acetyl) acrylohydrazide (Compound 13)
Synthesis of 2,2'-Azandiyl dipropane-1-ol:
2-Aminopropane-1-ol (5 g, 66.57 mmol) and 1-hydroxypropan-2-one (5.77 g, 77.89 mmol) were dissolved in ethanol (115 mL) and 50 mg of PtO ₂ was added. The reaction mixture was stirred at room temperature 50 psi H ₂ for 24 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the crude product, which was used in the next step without further purification. Crude yield 79%. ^{1 1} H NMR (400 MHz, CDCl ₃ ) δ 4.45 (bs, 2H), 3.42-3.43 (m, 1H), 3.16-3.22 (m, 4H), 2.65- 2.69 (m, 2H) 0.87-0.91 (m, 6H): LCMS m / z 133.99 [M + H] ⁺ , t _R : 4.077 min.

Synthesis of 3,5-dimethylmorpholine:
2,2'-Azandiyldipropane-1-ol (7 g, 52 mmol) was suspended in concentrated H ₂ SO ₄ (5.3 mL, 99.8 mmol) at room temperature and heated at 180 ° C. for 8 hours. The reaction mixture was cooled to 0 ° C. and a solution of KOH (11.79 g, 21.02 mmol) in 60 mL water was added dropwise. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered and the filtrate was extracted with CHCl ₃ : MeOH (85:15; 5 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 3.5 g of crude product, which was used in the next step without further purification (crude yield: 58%). ).

Synthesis of ethyl 2- (3,5-dimethylmorpholino) acetic acid:
Potassium carbonate (0.311 g, 2.25 mmol) and ethyl bromoacetate (0.319 g, 1.91 mmol) in a solution of 3,5-dimethylmorpholine (0.2 g, 1.73 mmol) in acetonitrile (4 mL) at room temperature. Added. The reaction mixture was stirred at 60 ° C. for 12 hours. The reaction mixture was transferred to ice water and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give the crude product, which was used in the next step without further purification (crude yield). : 54%).

Synthesis of Ethyl 2- (3,5-Dimethylmorpholino) Acethydrazide:
Ethyl-2- (3,5-dimethylmorpholino) acetic acid (0.19 g, 0.944 mmol) was dissolved in ethanol (4 mL), and d-hydrazine hydrate (0.047 g, 0.944 mmol) was added dropwise. did. The reaction mixture was stirred at 80 ° C. for 20 hours and the reaction mixture was concentrated under reduced pressure to give the crude product, which was used in subsequent steps without further purification. Crude yield 97%. ^{1 1} H NMR (400 MHz, DMSO-d ₆ ) δ 8.95 (s, 2H), 8.84 (s, 1H), 3.60-3.63 (m, 2H), 3.25-3. 29 (m, 2H), 3.14 (s, 2H), 3.05 (s, 2H), 0.86-0.88 (m, 6H): LCMS m / z 188.12 [M + H] ⁺ , t _R 4.716 min.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (3,5-dimethyl) Morphorino) Acetyl) Synthesis of Acrylohydrazide:
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in THF (10 mL) (0.2 g, To a solution of 0.569 mmol) and 2- (3,5-dimethylmorpholino) acetohydrazide (0.106 g, 0.569 mmol), T3P (0.543 g, 0.854 mmol) followed by DIPEA (0.110 g). , 0.854 mmol) was added at −60 ° C. and the mixture was stirred for 2 hours. The reaction mixture was transferred to 25 mL of ice water, extracted with ethyl acetate (2 x 25 mL), the combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to concentrate the crude product. Obtained and purified by chromatography (0-3% MeOH / CH ₂ Cl ₂ ) to 0.02 g of (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) phenyl). ) -1H-1,2,4-triazole-1-yl) -N'-(2- (3,5-dimethylmorpholino) acetyl) acrylohydrazide (yield: 7%) was obtained. ^{1 1} H NMR (400 MHz, DMSO-d ₆ ) δ 10.58 (s, 1H), 9.83 (s, 1H), 9.56 (s, 1H), 8.54-8.56 (m, 2H), 8.25-8.30 (m, 1H), 7.49-7.51 (d, J = 10.4 Hz, 1H)), 6.01-6.04 (d, J = 10) .4 Hz, 1H), 3.44-3.57 (m, 2H), 3.28-3.34 (m, 2H), 3.21 (s, 1H), 3.15 (s, 1H) , 2.84-2.88 (m, 2H), 0.93-1.04 (m, 6H): LCMS m / z 521.18 [M + H] ⁺ , t _R 1.898 min.

Example 14. (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (3-oxomorpholino) Synthesis of acetyl) acrylohydrazide (Compound 14)
Synthesis of ethyl 2- (3-oxomorpholino) acetic acid:
Morpholine-3-one (3 g, 29.67 mmol) was dissolved in DMF (15 mL, 29.67 mmol) and NaH (1.78 g, 44.51 mmol) was added at 0 ° C. The reaction mixture was stirred at room temperature for 30 minutes and ethyl bromoacetate (3.76 mL, 32.64 mmol) was added dropwise. The reaction mixture was further stirred at room temperature for 3 hours, transferred to 50 mL of water and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (2 x 50 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product, which was chromatographed (0-100% ethyl acetate / hexane). To obtain 600 mg of ethyl-2- (3-oxomorpholino) acetic acid (yield: 10%). LCMS m / z 187 [M + H]^＋, T_R 2.505 min.

Synthesis of 2- (3-oxomorpholino) acetohydrazide:
Ethyl-2- (3-oxomorpholino) acetic acid (600 mg, 3.21 mmol) was dissolved in ethanol (3 mL) and hydrazine hydrate (160.46 mg, 3.21 mmol) was added at room temperature. The reaction mixture was heated at 80 ° C. for 1 hour. The reaction mixture was transferred to 50 mL of water and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product, which was used in subsequent steps without further purification (crude yield: 54). %). LCMS m / z 174.05 [M + H] ⁺ t _R 2.489 min.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (3-oxomorpholino) Acetyl) Synthesis of acrylohydrazide:
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid (0.400 g, 1.14 mmol) in THF Dissolved in (4 mL), 2- (3-oxomorpholino) acetohydrazide (0.295 g, 1.71 mmol) was added. T ₃ P (1.09 g, 1.71 mmol) followed by DIPEA (220.80 mg, 1.71 mmol) was added dropwise at 60 ° C. and the reaction mixture was stirred for 1 hour. The reaction mixture was transferred to 25 mL of ice water and extracted with EtOAc (2 x 25 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure to give a crude product, purified by chromatography (0-4% MeOH / CH ₂ Cl ₂ ), and 0. .05 g of (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (3- (3-) 3- Oxomorpholino) acetyl) acrylohydrazide (yield: 8%) was obtained. ^{1 1} H NMR (400 MHz, DMSO-d ₆ ) δ 10.33 (bs, 2H), 9.63 (s, 1H), 8.57 (s, 2H), 8.30 (s, 1H), 7 .50-7.52 (d, J = 8 Hz, 1H)), 6.01-6.03 (d, J = 8 Hz, 1H), 4.08-4.12 (m, 4H), 3 .85-3.87 (m, 2H), 3.41-3.44 (m, 2H). LCMS m / z 507.13 [M + H] ⁺ , t _R 1.950 min.

Example 15. (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (3,3-dimethyl) Morphorino) Acetyl) Synthesis of Acrylohydrazide (Compound 15)
Synthesis of ethyl 2- (3,3-dimethylmorpholino) acetic acid:
3,3-Dimethylmorpholine (1 g, 8.68 mmol) was dissolved in acetonitrile (5 mL) and potassium carbonate (1.8 g, 13 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes and ethyl bromoacetate (1.1 mL, 9.55 mmol) was added. The reaction mixture was heated at 60 ° C. for 1 hour. The reaction mixture was then transferred to 50 mL of water and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product, which was used in the next step without further purification (crude yield:). 91%). LCMS m / z 202.9 [M + H] ⁺ , t _R 2.33 min.

Synthesis of 2- (3,3-dimethylmorpholino) acetohydrazide:
Hydrazine hydrate (0.20 mL, 2.98 mmol) was added to a solution of ethyl 2- (3-oxomorpholino) acetic acid (600 mg, 2.98 mmol) in tanol (3 mL) at room temperature. The reaction mixture was heated at 80 ° C. for 1 hour, allowed to cool to room temperature, transferred to 50 mL of water and extracted with ethyl acetate (3 x 25 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product, which was used in subsequent steps without further purification (crude yield: 28). %). LCMS m / z 188 [M + H] ⁺ t _R : 188 min.

(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (3,3-dimethyl) Morphorino) Acetyl) Synthesis of Acrylohydrazide:
(Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) acrylic acid in THF (2.5 mL) (0. In a solution of 250 g, 0.7 mmol) and 2- (3,3-dimethylmorpholino) acetohydrazide (0.160 g, 0.85 mmol), T ₃ P (0.63 mL, 1.06 mmol) followed by DIPEA ( 0.18 mL, 1.06 mmol) was added dropwise at −60 ° C. The reaction mixture was stirred for 1 hour, transferred to 25 mL of ice water and extracted with ethyl acetate (2 x 25 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure to give a crude product, purified by chromatography (0-4% MeOH / CH ₂ Cl ₂ ), and 0. .05 g of (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole-1-yl) -N'-(2- (3,3) 3-Dimethylmorpholino) acetyl) acrylohydrazide (yield: 13%) was obtained. ^{1 1} H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.81 (s, 1H), 9.62 (s, 1H), 8.56 (s, 2H), 8. 29 (s, 1H), 7.49-7.51 (d, J = 10.4 Hz, 1H)), 6.01-6.03 (d, J = 10.4 Hz, 1H), 3. 65-3.67 (m, 2H), 3.30-3.34 (m, 2H), 3.08 (bs, 2H), 2.55-2.58 (m, 2H), 0.96 ( s, 6H). LCMS m / z 521.18 [M + H] ⁺ , tR 1.937 min.

Example 16. Assays Compounds of a particular invention were tested in various assays, along with compounds X-1, X-2, and X-3 (shown below).

Inhibition of Nuclear Exports Inhibition of CRM1-mediated nuclear exports by the compounds of the invention was determined. The results are shown in Table 2. The inhibitory activity of the compound against the CRM1 protein was determined by the RevGFP assay. Compounds of the invention are active at _{an IC 50} of <10 [mu] M in Rev-GFP assay, the most preferred compounds have an activity _{IC 50} values of 1 [mu] M.

Experimental Protocol: Rev is a protein from human immunodeficiency virus type 1 (HIV-1) that has a nuclear export signal (NES) in its C-terminal domain and a nuclear localization signal (NLS) in its N-terminal region. contains. Nuclear exports of Rev proteins depend on the classical NES / CRM1 pathway (Nevile et al. 1997, Kau et al. 2003). Rev nuclear accumulation is observed in cells treated with a specific inhibitor of CRM1 such as LMB (Kau et al. 2003). In this assay, U2OS-RevGFP cells are inoculated on a 384-well black clear bottom plate the day before the experiment. Starting from 40 μM in separate 384-well plates, the compounds are serially diluted 1: 2 in DMEM and then transferred onto cells. The cells are incubated with the compound for about 1 hour, then fixed with 3.7% formaldehyde and nuclear stained with Hoechst 33258. By measuring the GFP amount of cells in the nucleus, it was determined _{IC 50} of compound (Kau et al.2003).

MTT Cell Proliferation Assay CellTiter 96® AQueuus One Solution Cell Proliferation Assay (Promega) was performed by MM. It was used in 1S, Jurkat and HCT-116 cells to test the cytotoxicity and cell division inhibitory properties of the compounds. The assay is based on cleavage of the tetrazolium salt, MTS, in the presence of the electron binding reagent PES (phenazine ethosulfate). The MTS tetrazolium compound is bioreduced by cells to a colored formazan product, which is soluble in tissue culture. This conversion is probably accomplished by NADPH or NADH produced by the metabolically active cellular dehydrogenase enzyme. The assay is performed by adding a small amount of CellTiter 96® AQueuus One solution reagent directly to the culture wells, incubating for 1-4 hours, and then reading the absorbance at 490 nm using a 96-well plate reader. Absorbance revealed that cell number and their metabolic activity were directly correlated. Cells were inoculated with 5 × 10 ^{3 to} 1.5 × 10 ⁴ cells (depending on cell type) in 100 μL of fresh culture in each well of a 96-well plate to inoculate one attached cell. It was attached at night. The stock solution of the compound was diluted with cell culture medium to give each drug in eight concentrations ranging from 1 nM to 30 μM, and DMSO <1% v / v was used as the negative control. After 72 hours of treatment, 20 μl of CellTiter 96® AQueuous reagent was added to each well of the 96-well assay plate and the plates were cultured in a humidified 5% CO ₂ atmosphere at 37 ° C. for 1-4 hours. The absorbance of each well was then recorded at 490 nm using a 96-well plate reader. In most cases, the assay was performed in triplicate and the results were presented as the maximum half-inhibition concentration (IC ₅₀ ) described below. In contrast to compound concentration was plotted the optical density was analyzed using non-linear regression equation (Excelfit), IC ₅₀ was calculated for each compound. The results are shown in Table 2.

Pharmacokinetics (PK) and brain: Plasma ratio determination Blood was collected from mice (N = 3) and at a total of 10 time points (before administration, 5 minutes after administration, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours). , 8 hours, 12 hours, and 24 hours). Mice were alternately drawn and each mouse provided blood draws at three time points. At the specified time points, animals were anesthetized under isoflurane by retro-orbital puncture, were taken about 110μL of blood per time point, were placed in a K ₂ EDTA (anticoagulant) tube prechilled. Blood samples were placed on wet ice and centrifuged (2000 g, 4 ° C. for 5 minutes) within 30 minutes of sampling to obtain plasma. All samples were stored frozen at about −80 ° C. until analysis. Prior to analysis, the sample was mixed with an internal standard (dexamethasone) in acetonitrile, vortexed, centrifuged and the supernatant injected for analysis. The concentration of the compound in plasma was measured using an LC-MS-MS measuring means (API 4000, triple quadrupole by electrospray ionization; Acity Ultra Performance Liquid Chromatography column C18, MeOH and formic acid as organic solvents). WinNonlin Professional 6.2 software package, using the non-compartmental pharmacokinetic model NCA200, including, but not limited to, Tmax, Cmax, t _1/2 , AUC _last , AUC _inf PK parameters Was calculated.

Brain-to-plasma ratio (B: P)
Separate mouse groups (N = 3) were dosed (10 mg / kg orally unless otherwise noted) and then killed at maximum plasma concentration (estimated T _max 2 hours post-dose) to final plasma and The brain was collected. Following collection, brain tissue was washed with cold saline, dried on filter paper, weighed, placed on dry ice and snap frozen. All samples were stored frozen at about −80 ° C. until analysis. During analysis, compounds that homogenize brain tissue (homogenized solution PBS, pH 7.4), mix with internal standard (dexamethasone) in acetonitrile, vortex, centrifuge, and use LC-MS-MS procedure. The supernatant was injected for concentration analysis (API 4000, triple quadrupole by electrospray ionization; Acetonitrile Performance Liquid Chromatografy column C18, MeOH and formic acid as organic solvents). Plasma samples were treated in the same manner (except for the homogenization step) and compound concentrations in either matrix were calculated based on the standard curve created. The results are shown in Table 2.

The AUC _Inf of compound X-1 was below the detection limit when administered orally at 10 mg / kg to mice. When administered intravenously at 5 mg / kg, Compound X-1 showed minimal exposure, as indicated by the low AUC _Inf of 209 hr · ng / mL. The brain-to-plasma ratio of compound X-1 was not determined due to its negligible intracerebral levels (below the limit of quantification) upon oral administration.

The AUC _Inf of compound X-2 was calculated to be 68.3 hr · ng / mL when 10 mg / kg was orally administered to rats. Such exposure levels are extremely low compared to compound X-3 and the compounds of formula I of the present invention. However, compound X-2 exhibits a moderate brain: plasma ratio. A low AUC _Inf , coupled with a non-negligible brain: plasma ratio, suggests that compound X-2 can cross the BBB despite low exposure levels. Applicants believe that compound X-2 would have had a significantly higher brain: plasma ratio if its AUC _Inf increased.

The AUC _Inf of compound X-3 was calculated to be 12300 hr · ng / mL when orally administered to rats at 10 mg / kg, indicating good exposure. However, X-3 demonstrated a high B: P ratio of 5.0.

All compounds of formula I show high AUC _Inf (> 3500 hr · ng / mL) and relatively low B: P (<2.5). In general, higher exposure levels of therapeutic agents often increase the likelihood of brain penetration. It is therefore surprising and surprising that the compounds of formula I exhibit relatively low brain: plasma ratios while exhibiting high AUC _Inf levels.

Example 17. Evaluation of Effect of Compound 2 on Tumor Growth of Z-138 Lymphoma Cell Lines Proliferated as Xenografts in Model SCID Mice Z-138 (ATCC # CRL-3001) Mantle Cell Lymphoma Cells were obtained from ATCC. These cells were cultured in IMEM medium supplemented with 10% horse serum, 1% penicillin and streptomycin, and 2 mM L-glutamine. The cells were diluted at a ratio of 1: 5 to 1:10 and secondarily cultured. Twenty-four 5- to 6-week-old female CB-17 SCID mice (Charles River Laboratories strain code 236) were used. A 0.2 mL volume of Z-138 cells, equivalent to 4 × 10 ⁷ cells per mouse, was inoculated into the left abdomen of SCID mice.

Treatment was initiated when the tumor reached an average volume of 84.3 mm ³ . Prior to the start of treatment, 8 mice were assigned to 4 groups based on tumor volume so that the average tumor volume in each group was within the range of 77-92 mm ³ . Mice were treated with vehicle, standard therapeutic agent / positive control agent (cyclophosphamide) or compound 2, as shown in Table 3.

Animals were allowed free intake of Labdiet® 5001 rodent solid feed and sterile water. Tumors were measured with a microcaliper once every two days and the tumor volume was calculated as (length x width x width) / 2. All animals were weighed daily to assess possible differences in body weight within the treatment group as an indicator of possible toxicity due to treatment. Animals that lost more than 20% of their starting weight were euthanized. Mice with weight loss greater than 15% of their starting body weight were not retreated until the weight loss recovered to less than 5% of their starting body weight. All animals with tumor volumes greater than 1500 mm ³ were euthanized.

The dosing solution was prepared fresh on each dosing day. Compound 2 was provided as a lyophilized powder containing 69.61% of Compound 2 and the rest consisting of Pluronic F-68 and PVP K29 / 32. It was prepared by dissolving the lyophilized powder in sterile water. Cyclophosphamide was dissolved in sterile injectable water at 8 mg / mL. All test substances were administered at a volume of 10 mL / kg body weight.

Statistical differences between treatment groups were determined using the Mann-Whitney rank sum or ANOVA test with a rejection limit of 0.05.

FIG. 1 shows a statistically significant reduction in tumor growth compared to vehicle when evaluated by comparing the area under the growth curve using the ANOVA test for both tumor volume and tumor volume percentage. All treatment groups are shown. These treatment groups showed a significant decrease in tumor growth at p <0.0001. Some weight loss was observed in the group treated with compound 2 at 15 mg / kg, and although statistically significant, severe weight loss was limited to a small number of animals compared to vehicle controls.

Orally administered Compound 2 had a dose-dependent antitumor effect at both 7.5 mg / kg and 15 mg / kg administrations.

Antitumor Activity of Compound 2 in A549 Small Cell Lung Cancer Model The A549 cell line was derived from alveolar carcinoma tissue explant culture in a 58 year old white male. Cells were cultured in ham F12-K tissue medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Cells were routinely trypsinized and passaged at 1:10. Thirty-two 5- to 6-week-old female CB-17SCID mice (Charles River Laboratories lineage code 236) with an average pretreatment weight of 16.3 grams were used. Prior to the start of treatment, 8 mice were divided into 4 groups based on tumor volume. Day of transplantation, the cells were washed with PBS, trypsinized, re-suspended in complete medium at a density of 2 × 10 ⁷ cells / mL, was mixed with matrigel equal volume. Mice were then subcutaneously inoculated with a volume of 0.1 mL using a 23 G needle.

Mice were treated with vehicle, standard therapeutic agent / positive control agent (cisplatin) or compound 2, as shown in Table 4. Animal weight and condition were recorded daily, tumors were measured with microcalipers on Mondays, Wednesdays and Fridays and the tumor volume was calculated as (length x width x width) / 2.

Animals that lost more than 20% of their starting weight were euthanized. Mice with weight loss greater than 15% of their starting body weight were not retreated until the weight loss recovered to less than 5% of their starting body weight. All animals with tumor volumes greater than 1500 mm ³ were euthanized.

The dosing solution was prepared fresh on each dosing day. Compound 2 was provided as a lyophilized powder containing 69.61% of Compound 2 and the rest consisting of Pluronic F-68 and PVP K29 / 32. It was prepared by dissolving the lyophilized powder in sterile water. Cisplatin was dissolved in DMSO at 5 mg / mL and diluted 1:10 in sterile injectable water. All test substances were administered at a volume of 0.1 mL / g body weight.

Statistical differences between treatment groups were determined using the Mann-Whitney rank sum or ANOVA test with a rejection limit of 0.05.

Data on tumor volume changes during the study are shown in FIG. Mean tumor volume of the vehicle control group increased from Day 1 95 mm ³ to 29 days of 1669mm ^3. Cisplatin treatment group had a mean tumor volume of 104 mm ³ on day 1, was increased to 1136Mm ³ to 29 days. Mice treated with 10 mg / kg oral Compound 2 (Group 3) had an average tumor volume of 101 mm ³ on day 1, which increased to 686 mm ³ by day 29. 5 mg / kg orally mice treated with compound 2 (group 6) has an average tumor volume of 101 mm ³ on day 1, it was increased to 1231Mm ³ to day 29.

Additional analysis of tumor volume data was performed by calculating the area under the mean curve (AUC) of each tumor and comparing the groups using a centralized ANOVA test. This analysis suggested that there was a statistically significant difference (p = 0.0005) between the vehicle control group and the group treated with 10 mg / kg of Compound 2. It should be noted that there was no statistically significant reduction in tumor growth in the positive control group (cisplatin).

Orally administered Compound 2 had a dose-dependent antitumor effect at both 5 mg / kg and 10 mg / kg administrations. However, only the 10 mg / kg group showed a statistically significant difference compared to the vehicle-treated group.

Evaluation of Compound 2 in Anti-Collagen Antibodies-Induced Rheumatoid Arthritis (CAIA) Mouse Model Twenty-four male Balb / c mice aged 6-8 weeks were used. Animal weight deviations at the start of treatment did not exceed ± 20% of mean body weight. Animals were randomly assigned to 3 groups receiving vehicle, dexamethasone or compound 2. On the 0th day of the test (test start date), 2 mg intravenous injection of ArtritoMAbTM antibody cocktail (MD Biosciences # S1203001) was performed in all mice, and the LPS (100 μg / mouse) was intraperitoneally injected on the 3rd day of the test. It followed. Test animals were treated with oral 7.5 mg / kg Compound 2 or 4 mg / kg Compound 2; intraperitoneally 1 mg / kg dexamethasone; or oral vehicle. The therapeutic agent is administered once daily for 4, 6, 8, and 10 days in all groups, except where a washout period applies. If the animal's body weight dropped to less than 87%, the animal was not dosed until it gained more than 90% of its day 0 body weight.

On days 0, 3-8, 10 and 12 of the study, all mice were monitored for arthritis onset, clinical signs, and body weight. Observations included changes in the skin, fur, eyes, mucous membranes; appearance of secretions and excreta (eg, diarrhea); and autonomic activity (eg, lacrimation, salivation, naps, pupil size, abnormal respiratory patterns). All paws (left and right forefoot, and left and right posterior) of each animal on day 0 of the study and before administration of the test or control, followed by days 3-8, 10, and 12 (end of study) of the study. Feet) were examined for arthritis-induced response signs. Arthritic responses were scored and recorded according to a scale of 0-4 in ascending order of severity, as shown in Table 5 below. Foot thickness was also assessed using dial calipers (Kroeplin, Munich, Germany).

Dosages were calculated based on the hypothesis that animals weigh an average of 20 g. The stock solution of dexamethasone was prepared in 100% ethanol and diluted with PBS to the appropriate concentration before use. Vehicles for the vehicle control group were prepared by dissolving 0.6 g of Pluronic and 0.6 g of PVP in 100 mL of distilled deionized water. The MAb stock solution (10 mg / mL) was supplied from MD Biosciences, Division of Morwell Diagnostics GmbH. LPS was diluted with PBS to the appropriate concentration. Immediately before the injection, a thorough vortex was needed. Compound 2 was provided as a lyophilized drug powder containing 70.71% of Compound 2 and the rest consisting of Pluronic F-68 and PVP K29 / 32. A fixed volume of 200 μL was administered to each mouse.

The assessment was based primarily on the average of arthritis scores and foot thickness measurements. Where appropriate, ANOVA data analysis with a Chuky post-test was applied to determine the significance of the therapeutic effect.

3A and 3B show the results of the CAIA mouse model experiment. Following LPS booster immunization on day 3, clinical signs associated with LPS-administration developed in all groups. Compared to vehicle-treated mice, mice treated with 7.5 mg / kg or 4 mg / kg of Compound 2 had significantly lower total arthritis scores on days 5-12 and 6-12, respectively. Dexamethasone treatment significantly reduced the total arthritis score on days 6-12 compared to the vehicle group. Mice treated with 7.5 mg / kg or 4 mg / kg of Compound 2 had a significantly reduced hindlimb arthritis score on days 5-12 as compared to vehicle-treated mice. Dexamethasone treatment significantly reduced hindfoot arthritis scores on days 5 and 12 compared to the vehicle group. There was no significant difference in body weight between the vehicle-treated group and the test-treated group.

Considering the findings of this study, Orally delivered 7.5 mg / kg or 4 mg / kg of Compound 2 reduced the persistence of mean arthritis score and foot thickness in an anti-collagen antibody-induced model of rheumatoid arthritis. Significant anti-arthritis activity was shown with a decrease in.

Efficacy Test of Compound 2 for Collagen-Induced Arthritis (CIA) in Lewis Rats 10 female Lewis rats (BK) 6-8 weeks old, each weighing 180-200 g before treatment It was randomly divided into 4 groups (groups A to D). Rats in groups B to D were intradermally immunized with bovine CII in IFA at 3 sites on the back near the base of the tail with 500 μL emulsion (200 μL, 200 μL, 100 μL for each site) on day 0. .. On day 7, rats in groups B to D were boosted with the same amount of emulsion intradermally near the previous injection site. Treatment In treatment models (groups C and D), dexamethasone or compound 2 was orally administered to rats with CIA after the onset of arthritis, as shown in Table 6. Rats were weighed daily and animals were given drug holidays if they lost more than 13%.

CIA onset was assessed by macroscopic scoring and foot swelling measurements. This was assessed daily for the first 5 days after sensitization (7th day) and twice a week for the rest (Monday and Thursday) by the clinical scoring system for each paw shown in Table 7.

Foot volume was measured by volumetric method on the same day as the arthritis measurement throughout the study period. The volume and swelling ratio of each hind paw was measured and the following formula was used.
Swelling ratio _{= (C N -C 0) /} C 0 × 100%

FIG. 4A is a graph of joint swelling over time, showing joint swelling measured on a scale of 0-4 in unsensitized rats and rats treated according to the model with positive controls or Compound 2.

4 mg / mL bovine CII (in 10 mM acetic acid) was emulsified with an equal volume of FA.

The clinical scores were totaled for each animal and the overall mean of all animals in each group was expressed as the mean arthritis score. FIG. 4B is a graph of clinical scores as a function of time, showing unsensitized rats and rat arthritis scores treated according to the model with positive controls or Compound 2.

On day 28 of the study, 3 animals representing each treatment group were euthanized, hind paws were harvested and stored in 4% neutral buffered formalin. The prepared hind paw sections were stained with hematoxylin and eosin (H & E).

Histopathological analysis of control animals showed cartilage erosion and pannus formation consistent with the disease course. However, in rats treated with Compound 2, relatively intact cartilage was found on the joint surface and pannus formation was minimal. The results of histological analysis are shown in FIG.

Laboratory scores, joint swelling, and histological examination data showed a correlation. The results also showed the therapeutic efficacy of Compound 2 at 4 mg / kg (MPK), as indicated by its effect on clinical scores, joint swelling, and histological examination. The results of the CIA model in Lewis rats are depicted in FIGS. 4A and 4B, and FIG.

Anti-psoriasis activity of Compound 2 against phorbol-12-millistate-13-acetic acid (PMA) -induced psoriasis in female BALB / C mice Using 24 6-8 week old female BALB / c mice weighing 22-30 g did. Mice were randomized into 4 groups of 8 mice each. The groups of animals were as follows: Group I (unsensitized; ethanol), Group II (PMA; ethanol), Group III (PMA; 10 μM compound 2), and Group IV (PMA; betamethasone). .. 20 μL of PMA (4 μg / 20 μL of acetone) was topically applied to the upper surface of the pinna of all animals in groups II-IV. PMA was applied daily to the left ear and every other day (Monday, Wednesday and Friday) to the right ear on days 1-9. Thirty minutes after application of PMA, vehicle or standard compound (betamethasone) or compound 2 was applied topically to the ears of different groups of animals. Note that on days 1-12, the vehicle, standard compound, and compound 2 were applied daily to both ears of different animals.

Animals were observed daily for 12 days for all treatment-related symptoms. Basal pinna thickness was recorded in all animals (prior to PMA application) using a digital screw gauge at T0 hours (day 1). Throughout the duration of the test, 4 hours after application of vehicle, standard compound, or compound 2, ear thickness was measured daily using a digital screw gauge and erythema, scales, and wrinkle cores were recorded. .. The severity of auricular injury was assessed by the scoring system shown in Table 8.

Animals were allowed free intake of a nutritionally balanced high-pressure steam-sterilized pellet diet (Nurivet Life Sciences, Pune (India)) throughout the duration of the experiment to access normal drinking water.

Formulations were prepared using commercially available 100% DMSO (LR grade) and ethanol (LR grade). PMA was prepared by dissolving 10 mg of PMA in 50.0 mL of acetone. Compound 2 was prepared by dissolving 1.47 mg of Compound 2 in 300 μL of 100% DMSO.

Experimental results are shown in FIGS. 6A-6D as mean ± SEM. There were no significant differences between all treatment groups in terms of body weight, food, and water consumption. PMA application was (i) increased thickness of the left and right ears (group II vs. insensitive), and (ii) increased disease activity index (DAI) of the left and right ears (group II vs. insensitive). )showed that. Importantly, topical application of Compound 2 resulted in a significant reduction in (i) left and right auricular thickness and (ii) PMA-induced increase in DAI in the left and right ears. This effect was significant on days 6-8 of the study, with more animals treated with Compound 2 having reduced left / right pinna thickness (compared to group II animals) and DAI (group II). Compared to animals in). It should be noted that the compound 2 mediated reduction in PMA-induced increase in left / right pinna and DAI decreased as the study progressed (after day 10).

In a PMA-induced psoriasis model in mice, Compound 2 showed statistically significant anti-psoriasis activity.

Anti-psoriasis activity of Compound 2 in an imiquimod (IMQ) -induced skin inflammation / psoriasis model (Test 1)
40 male BALB / c mice aged 6-8 weeks with a pretreatment weight of 22-30 g were used. BALB / c mice were randomized into groups 4) with 10 mice per group. A small area of dorsal skin (approximately 2 x 2 cm ² ) of all animals was shaved cleanly. Group I animals played the role of insensitive agents. From days 1 to 13, 31.25 mg of IMQ cream was topically applied daily to the dorsal side of the animals to group II-IV [Group II (IMQ; vehicle), Group III (IMQ; Compound 2 (1 μM)), And group IV (IMQ; cyclophosphamide (10 mg / kg)] induced psoriasis. Daily from day 1 to 13 hours, 4 hours after application of IMQ, vehicle or standard compound (cyclophosphamide) or compound 2. Was administered to the appropriate group (locally 30 μL; orally according to body weight). 2 hours after administration of the vehicle or standard compound or compound 2, skin psoriasis, scales, wrinkles, and thickening were recorded. , Disease activity index (DAI) was determined.

Animals were observed daily for 13 days for all treatment-related symptoms. Daily observations include body weight, feed intake, skin thickening, scales, wrinkles, erythema, nasal juice, movement, breathing, hair, bulging abdomen, skin condition, fur, mucous membranes, presence or absence of secretions, eye condition, tail Included elevation, athletic performance, posture, and walking. The severity of dorsal skin injury was assessed by assigning erythema, scales, wrinkles, and skin thickening scores based on visual observations according to the skin items in Table 9.

Formulations were prepared using commercially available 100% DMSO (LR grade), ethanol (LR grade), cyclophosphamide (CMC), PVP, and Pluronic. Compound 2 was prepared by dissolving 1.47 mg of Compound 2 in 300 μL of 100% DMSO. Cyclophosphamide was prepared by dissolving 500 mg of CMC in 100 mL of distilled water.

The experimental results shown in FIGS. 7A and 7B are expressed as mean ± SEM.

During the duration of the study, there were no significant differences in body weight, food intake, and water intake in the treatment group compared to the control group. Compound 2 reduced the onset of symptoms of IMQ-induced disease.

Compound 2 exhibits anti-psoriasis activity, as evidenced by a reduced disease activity index compared to the vehicle-treated group. In addition, Compound 2 caused this effect without adversely affecting body weight, food and water intake.

Anti-psoriasis activity of Compound 2 in an imiquimod (IMQ) -induced skin inflammation / psoriasis model (Test 2)
Forty male BALB / c mice (Biological E Limited, Hyderabad (CPCSEA registration number: 36/99 / CPCSEA)) were divided into 4 groups consisting of 10 mice each. Animals were randomized based on their weight. The groups were Group I (unsensitized), Group II (IMQ; vehicle (PEG400 and HPBCD)), Group III (IMQ; Compound 2 (2.5 mg / kg)), and Group IX (IMQ; cyclophosphamide). (10 mg / kg))).

Small dorsal areas of each mouse were shaved to ensure that these areas were of comparable size / area. From days 1 to 6, 50 mg of IMQ cream was topically applied daily to the dorsal side of the animals to induce psoriasis in groups II-IV. On days 1 and 2 of the study, compound 2 or a positive control (cyclophosphamide) or vehicle was administered to a reasonable group of animals 4 hours after topical application of IMQ. It should be noted that Group II and Group III animals were injected subcutaneously, whereas Group IV animals were orally administered. Treatment with Compound 2, vehicle, and cyclophosphamide was completed on day 2. In these groups, daily IMQ treatment was continued until day 6. On day 7, psoriasis-inducing animals were again randomized into 3 groups of 10 animals each, based on the Cumulative Disease Activity Index (CDAI). Animals were administered vehicle or positive control or Compound 2 from day 7 to day 9. Note that during this time the animals were not treated with IMQ. From day 10 to day 14, animals were optionally treated with IMQ (days 10, 12, and 14), or vehicle, positive control or compound 2 (days 11, 13).

All animals were observed daily for 16 days for macroscopic observation, body weight and feed and water intake. On days 1 and 2, erythema, scales, wrinkles, and skin thickening scores were recorded 2 hours after vehicle / positive control / test compound administration, and on days 3-14, IMQ application or positive. Scores were recorded 4 hours after administration of control, vehicle or compound 2. The severity of induction on the dorsal side of the animal was assessed and scored as shown in Table 10.

The vehicle was prepared by dissolving 40 mg of HPBCD in 70.0 mL of distilled water. Compound 2 was prepared by dissolving 3.59 mg in 0.5% PVP and 0.5% pluronic. Cyclophosphamide was prepared by dissolving 500 mg of CMC in 100 mL of distilled water.

The experimental results shown in Table 10 are expressed as mean ± SEM. Data were evaluated using one-way ANOVA and post-test was performed using Dunnett's test.

It was observed that the rate of decrease in disease activity index in animals treated with Compound 2 was significantly greater than that observed in vehicle-treated animals. During the duration of the study, there were no significant differences in body weight, food intake, and water intake in the treatment group compared to the control group.

The results obtained showed that treatment with Compound 2 reduced the onset of symptoms of IMQ-induced disease without significantly affecting food or water intake and thus without showing any effect on animal body weight in the treatment group. Show that.

Effect of Compound 2 on Zucker Rats Twenty-one 7-month-old male Zucker rats were assigned to three groups with N = 7 based on equivalent body weight and food intake. As controls, an additional N = 7 group of Zucker lean controls of the same age was included. Body weight and food and water intake were assessed at approximately the same time each day (14:30 to 15:30 h). On the treatment day, the drug was administered from 14:30 to 15:30 h (about 2 hours before turning off the lights).

Zucker obese and lean controls were orally treated daily during weekdays with vehicle (10 mL / kg volume; 0.5% Pluronic F68 in water and 0.5% PVP K29 / 32). Both groups of Compound 2 (1.5 mg / kg and 3 mg / kg) were treated orally daily during weekdays (10 mL / kg dose volume; 0.5% Pluronic F68 and 0.5% PVP K29 in water). / 32). Prior to the treatment phase, 4-day baseline data was collected. The treatment phase lasted 16 days and also included 6 days of the washout phase.

8A and 8B and FIG. 9 show the effect of Compound 2 on Zucker rats. At baseline, there were no significant differences in body weight and daily food intake among the three Zucker obese groups. However, all groups were significantly different from the Zucker lean group.

Compound 2 (1.5-3 mg / kg orally) produced a dose-dependent reduction in daily food intake and body weight over a 16-day treatment period compared to the Zucker control group. Compound 2 treatment also significantly increased the water intake measured during the same period. There was a significant difference in body weight gain between the 3 mg / kg compound 2 group and the Zucker vehicle group. There was no significant difference in body weight gain between the 1.5 mg / kg Compound 2 treatment group and the Zucker vehicle group.

Compound 2 showed a dose-dependent lowering effect on daily food, with higher doses (3 mg / kg) being more effective than doses of 1.5 mg / kg. In addition, the 3 mg / kg compound 2 group showed lower weight gain compared to the Zucker control group.

Effect of Compound 2 on Diet-Induced Obesity Model Two-month-old male Sprague dolly rats were fed a high-fat diet (Research Diets Inc., product code D12492, 60% kcal% fat) for 3 months. A group of age-matched rats were fed a standard lab solid diet (LabDiet 5001, about 13% kcal% fat), and these animals served as controls for the DIO group.

At 4 months of age, after 2 months of high fat diet, all rats were assigned to 3 groups with N = 7 based on equivalent body weight and food intake. Body weight and food and water intake were measured at approximately the same time each day. On the day of treatment, the drug was administered about 2 hours before the lights were turned off.

The DIO control group was treated daily on weekdays with a vehicle (oral, 10 mL / kg dose volume; 0.5% Pluronic F68 in water and 0.5% PVP K29 / 32). Two groups of 1.5 mg / kg compounds were orally treated daily during weekdays throughout the treatment phase (dose 10 mL / kg dose volume; 0.5% Pluronic F68 in water and 0.5% PVP K29 / 32). .. Two groups of 3 mg / kg compounds (10 mL / kg dose volume; 0.5% Pluronic F68 and 0.5% PVP K29 / 32 in water) were given once daily for the first week and then 2 weeks for 2 weeks. It was treated orally once (Monday, Wednesday). During the 3rd and 4th weeks of treatment, treatment with 3 mg / kg of Compound 2 was continued twice weekly, with dosing on Monday and Thursday.

Baseline data for 3 days were collected prior to the treatment phase. The treatment phase lasted 4 weeks. A 10-day washout stage was also included.

Compound 2 was supplied in powder form. The test compound had an active percentage of 65.89%. The active percentage was adjusted using a BEW of 1.437 and prepared by dissolving in 0.5% w / v Pluronic F-68 and 0.5% w / v PVP K-29-32 vehicle solution. did. The vehicle solution was prepared weekly, while compound 2 was freshly prepared every two days and stored at + 4 ° C. Animals were dosed with a volume of 10 mL / kg. Individual doses were calculated based on the latest body weight and provided appropriate mg / kg / day doses.

10A and 10B and FIG. 11 show the effect of Compound 2 on a diet-induced obesity model. At baseline, there were no significant differences in body weight and daily food and water intake among the three DIO groups. However, all DIO groups were significantly different from the normal diet group. In particular, animals fed a normal diet were significantly lighter in body weight than rats fed a high-fat diet. Conversely, rats fed a high-fat diet consumed significantly less food and water daily than rats fed a normal diet.

Compound 2 (1.5-3 mg / kg orally) produced a dose-dependent reduction in daily food intake and body weight over a 28-day treatment period compared to the DIO control group. Compound 2 treatment also significantly increased the water intake measured during the same period. (F3, 27 = 11.2, P <0.01).

Regarding the effect of treatment on weight gain, this was formally evaluated as a percentage of weight change from day 3 of the study. On days 7 (10th test) and 14th (17th test) of treatment, there was a significant reduction in weight gain in both compound 2 groups compared to the DIO control.

Body weight and food / water intake were measured daily during the washout phase. Food intake in the compound 2 group was similar to that of the DIO control. The body weight of the compound 2 group remained lower than that of the DIO control.

Compound 2 reduces daily food intake in a dose-dependent manner. Compound 2 also affects weight gain at both 1.5 and 3 mg / kg doses.

Induction of Nrf2 Anti-inflammatory Pathway of Compound 1 THP-1 (Human Acute Monocytic Leukemia Cell) cells were used to evaluate the effect of Compound 1 on the Nrf2 pathway in an inflammatory environment. Nuclear factor (red blood cell origin 2) -like 2 (Nrf2) is an anti-inflammatory transcription factor. Under normal conditions, Nrf2 is retained in the cytoplasm by Kelch-like ECH-binding protein 1 (KEAP1), which degrades Nrf2 by ubiquitination. Nrf2 can also migrate into the nucleus as a CRM1 cargo and return to the cytoplasm. In this study, Nrf2 is protected from degradation by knockdown of KEAP1 by siRNA. KEAP1-depleted cells were then treated with TNFα to test the ability of Compound 1 to induce inflammation and reverse inflammation by upregulation of the Nrf2 pathway. To demonstrate activation of the Nrf2 pathway, the expression of its two downstream genes, NAD (P) H dehydrolase [quinone] 1 (NQO1) and epoxide hydrolase 1 (EPHX1), was quantified by quantitative PCR.

THP-1 (acute) with RPMI-1640 medium (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and 2-mercaptoethanol in two 10 cm culture dishes (6 * 10 ⁶ cells / dish). Monocytic leukemia) cells were seeded to a final concentration of 0.05 mM. Lipofectamine RNA iMax (Invitrogen) was used to transfect cells in one culture dish with 50 nM KEAP1 siRNA (Life Technologies, Silencer Select, siRNA ID # s18982), while cells in another culture dish were transfected with 50 nM. Control siRNA, Block-iT (Invitrogen), was transfected. Transfected cells were allowed to stand for 72 hours before KEAP1 knockdown efficiency was calculated by quantitative PCR using a probe for KEAP1.

The cells from each culture dish were then equally divided into 4 wells in another 6-well plate. One of the wells from each plate was pretreated with 1 μM compound 1 for 1 hour, followed by 20 ng / mL TNFα for 24 hours. The other wells were treated with either 1 μM compound 1 or 20 ng / mL TNFα for 24 hours, or not with either. Following treatment, RNA was extracted from cells using an RNA extraction kit (Qiagen). RNA samples from each treatment group were reverse transcribed and real-time PCR was performed on the corresponding cDNA sequences using probes for Nrf2 and its two downstream genes NQO1 and EPHX1. THP-1 cells were transfected with KEAP1 siRNA. A knockdown efficiency of 40% was achieved. KEAP1 knockdown cells were treated with either 1 μM compound 1 and / or 20 ng / mL TNFα for 24 hours.

FIG. 12A shows a 2.5-fold increase in Nrf2 expression in cells treated with the combination of TNFα and compound 1 compared to untreated cells. However, similar (up to 3-fold) increases in Nrf2 mRNA levels are also found in cells treated with Compound 1 and TNFα without KEAP1 knockdown. Compound 1 or TNFα alone, with or without KEAP1 knockdown, had no significant effect on Nrf2 expression.

FIG. 12B shows the expression of NAD (P) H dehydrogenase [quinone] 1 or NQO1 in cells with or without KEAP1 knockdown. FIG. 12B shows that KEAP1 knockdown had an effect on NQO1 expression. The untreated sample showed a 2-fold increase in its mRNA level upon KEAP1 knockdown. The combination of Compound 1 and TNFα resulted in a 4-fold increase in NQO1 expression in the KEAP1 knockdown sample compared to the 2-fold increase seen in the same combination in cells without KEAP1 knockdown.

FIG. 12C shows the mRNA levels of epoxide hydrolase 1 or EPHX1 in cells with or without KEAP1 knockdown post-treatment with compound 1 and / or TNFα. FIG. 12C shows that Compound 1 upregulated the expression of EPHX1 in the presence or absence of TNFα. KEAP1 knockdown enhances the effect of compound 1 as up to 2.5-fold induction was observed in samples with compound 1 and KEAP1 knockdown.

Treatment with 1 μM compound 1 for 24 hours in the presence of 20 ng / mL TNFα upregulated Nrf2 signaling. KEAP1 knockdowns are seen in the higher NQO1 induction multiples (2-fold vs. 4-fold) and EPHX1 (1.5-fold vs. 2.5-fold) compared to their expression levels without KEAP1 knockdown. , This effect was enhanced. The results show that CRM1 inhibition can activate the Nrf2 pathway during inflammation, suggesting that treatment with compound 1 in combination with a KEAP1 inhibitor may be more effective than treatment with compound 1 alone. ..

Effect of Compounds 1, 2, and 12 on NF-κB transcriptional activity TNFα can induce transcriptional activity of NF-κB. This transcriptional activity is initiated when IκB, which binds to and inhibits NFκB, is degraded. The class II family member RelA or p65 of the NF-κB protein, which constitutes a heterodimer with the class I family member p50, then migrates into the nucleus. The p65 subunit has a transactivating domain at its C-terminus, which activates transcription of inflammation-related genes. Like NF-κB, IκB can also migrate into the cell nucleus. Nuclear accumulation of IκB protects proteins from degradation, as degradation occurs primarily in the cytoplasm. CRM1 is involved in nuclear exports of IκB. Blocking nuclear exports of IκB through inhibition of CRM minimizes NF-κB activity, as nuclear IκB binds to NF-κB and prevents NF-κB from binding to the DNA sequence.

Compounds were tested on HeLa (adenocarcinoma) cells to quantify their ability to inhibit NF-κB transcriptional activity. NF-κB activity was induced in HeLa cells by TNFα, and then compounds were added to inhibit inducible NF-κB activity. The maximum half-inhibitory concentration (IC ₅₀ ) of some compounds, compound 1, compound 2, and compound 12, was measured by dose response testing.

Eagle from Lonza seeded with HeLa cells in 12-well plates (200,000 cells / well) and supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and 50 μg / mL penicillin / streptomycin (Invitrogen). The cells were cultured in Minimal Essential Medium (EMEM) and left overnight for attachment. Cells were pretreated with serially diluted (starting at 30 μM; 1: 3 dilution) compound for 1 hour and then exposed to 20 ng / mL TNFα (Peprotech) in serum-free medium for 4 hours. After treatment, cells were washed with PBS (Invitrogen) and lysed in RIPA buffer (Themo Scientific). Transcriptional activity of NF-κB in cells was measured by a chemiluminescent transcription factor assay kit (Thermo Scientific; Catalog No. 89859) according to the manufacturer's instructions. Briefly, 1.5 mg / mL RIPA-dissolved whole cell extracts from each treatment were cultured in NF-κB biotinylated common sequence binding 96-well plates. The active NF-κB transcription factor bound to the common sequence was cultured with the NF-κB p65 primary antibody and then with the secondary HRP conjugated antibody. The signal obtained by adding the chemiluminescent substrate to the wells was detected using an illuminometer. For each concentration IC ₅₀ of curve it was analyzed separate experiments 3 times. Use the XLFit model 205, it was calculated the _{IC 50} curves.

Inhibition of NF-κB transcriptional activity was measured by 1 hour of pretreatment of the compound followed by 4 hours of exposure to 20 ng / mL TNFα followed by serial dilution of compound 1, compound 2 and compound 12. Three independent experiments are scored for each concentration and the averages are presented here. Compound 1 had an IC ₅₀ value of 1.59 μM, compound 2 had an IC ₅₀ value of 1.22 μM, and compound 12 had an IC ₅₀ value of 1.46 μM.

Evaluation of the effect of Compound 1 on the expression of the pro-inflammatory protein COX-2 in in vitro culture HeLa cells HeLa cells are cultured in 6 wells with EMEM medium (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen). They were seeded in dishes (2.5 × 10 ⁵ cells / well). Two of the wells of the plate were pretreated with 10 μM compound 1 for 30 minutes, at which time one of the wells was exposed to 20 ng / ml TNFα (Proprotech) for 1 hour. The other wells were treated with 20 ng / ml TNFα for 1 hour or no treatment. Following treatment, RNA was extracted from cells using an RNA extraction kit (Qiagen). RNA samples from each treatment group were reverse transcribed and quantitative real-time (qRT) PCR was performed on the corresponding cDNA sequences using probes for COX-2 (Life Technologies).

HeLa cells were seeded in 6-well culture dishes (5 × 10 ⁵ cells / well) with EMEM medium (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen). Two of the wells of the plate were pretreated with 1 μM compound 1 for 30 minutes, at which time one of the wells was exposed to 20 ng / ml TNFα (Proprotech) for 24 hours. The other wells were treated with 20 ng / ml TNFα for 24 hours or no treatment. Following treatment, the cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitor (Roche) to create whole cell lysates. Immunoblot detection of COX-2 protein was performed using an anti-COX-2 antibody (Cayman). For each sample, the signal intensity of the COX-2 protein was normalized for β-actin (Santa Cruz) and the graph was plotted as an arbitrary intensity unit.

Data from mRNA analysis by qRT-PCR are shown in FIG. 13A. One hour after treatment, TNFα induced an approximately 8-fold increase in COX-2 mRNA expression compared to controls, while compound 1 alone did not affect the level of COX-2 expression. .. Compound 1 did not cause increased COX-2 mRNA expression.

Data from protein analysis by immunoblot is shown in FIG. 13B. HeLa cells are left untreated or treated with either 20 ng / ml TNFα or 1 μM Compound 1 or treated with 20 ng / ml TNFα and 1 μM Compound 1 for 24 hours, followed by an immunoblot. The amount of COX-2 protein present was assessed by detection. COX-2 protein increased by 24 hours in TNFα-stimulated cells compared to untreated control and compound 1 treated cells, while compound 1 increased the amount of COX-2 protein in the presence of TNFα. Reduced. The immunoblot signal intensity of the COX-2 protein was normalized and graphed for β-actin in each sample.

Compound 1 does not affect TNFα-induced COX-2 expression, but reduces the amount of TNFα-induced COX-2 protein expression.

Compound 1 produces 4 HeLa and THP-1 (human acute monocytic leukemia) cells with the pro-inflammatory factor TNFα alone, which localizes to the nuclear-directed inflammation-related CRM1 cargo, or in combination with 1-10 μM compound 1. Treatment was performed for ~ 24 hours, and then the nuclear localization of inflammation-related CRM1 cargo proteins IκB, Nrf2, HMGB1, FoxP3, FOXO1a, RxRα, PPARγ, and NFκB (p65 subunit) was analyzed by immunofluorescence (IF).

To detect IkB, Nrf2, RxRα, and PPARγ localization, cells were precultured with 10 μM compound 1 for 30 minutes, followed by 4-hour culture with 20 ng / mL TNFα in serum-free medium. .. To detect HMGB1, FoxP3, and Fox1A, cells were pre-cultured with 1 μM Compound 1 for 2 hours, followed by a 24-hour culture with 20 ng / mL TNFα. To detect NFκB, cells were pre-cultured with 1 μM compound 1 for 2 hours, followed by a 24-hour culture with 20 ng / mL TNFα. Cells are fixed with 100% ice-cold methanol (Methanol) and permeabilized / blocked with 0.1% Tween 20, 0.3M glycine, and 1% BSA in PBS, or PFA (3% para in PBS). It was fixed with formaldehyde and 2% sucrose in) and permeabilized / blocked with 0.1% Triton-X100 and 1% BSA in PBS. IκB was detected by the primary rabbit monoclonal (E130) antibody (ab32518) from Abcam; Nrf2 was detected by the primary rabbit polyclonal antibody (sc722) from Santa Cruz; RxR alpha was the primary rabbit polyclonal antibody from Santa Cruz. Detected by (sc553); PPAR gamma was detected by primary rabbit monoclonal [E130] antibody (# 2443) from Cell Signaling; Foxo1A was detected by primary rabbit monoclonal [C29H4] antibody (# 2880) from Cell Signaling. HMGB1 was detected by the primary rabbit polyclonal antibody (ab18256) from Abcam; FoxP3 was detected by the primary rabbit polyclonal antibody (ab10563) from Abcam; NFκB-p65 was the primary rabbit from Cell Signaling. It was detected by a monoclonal [C22B4] antibody (# 4764). Rabbit secondary antibody Alexa Fluor 488 (Invitrogen, A11008) was used for all staining. The image was taken at 20x magnification.

Confinement of inflammation-related CRM1 cargo in the nucleus has an adverse effect on inflammation, so the IF assay can serve as a biomarker of the anti-inflammatory effect of CRMI inhibitors.

IκB is an pro-inflammatory pathway. It is an inhibitor of NFκB that induces the expression of NFκB. Since most IκB degradation occurs in the cytoplasm, its nuclear localization protects IκB from degradation and allows it to bind to nuclear NFκB, blocking the pro-inflammatory activity of NFκB. Nrf2 is a leucine zipper transcription factor that induces the expression of anti-inflammatory activity in the nucleus. HMGB1 is a high mobility group box 1 factor, which normally binds closely to chromatin. Upon active secretion or passive release from damaged cells, HMGB1 functions as a cytokine and induces an pro-inflammatory response. Confinement of HMGB1 in the nucleus interferes with its pro-inflammatory effect. FoxP3 (forkhead box P3) functions as a master transcription factor in the development and function of regulatory T cells with immunosuppressive activity. FOXO1a is a transcription factor capable of inducing anti-inflammatory genes such as angiopoetin-2. Therefore, nuclear localization protects FOXO1a from phosphorylation, nuclear elimination, and subsequent degradation. RxRα is a retinoid nuclear receptor that regulates the expression of chemokines such as Ccl6 and Ccl9 in macrophages. RxRα is essential for leukocyte recruitment to the site of inflammation. Encapsulation of RxRα results in the recruitment and depletion of transcriptional activation cofactors that would otherwise aid in the binding of pro-inflammatory transcription factors such as NFκB. PPARγ is a ligand-activated transcription factor belonging to the nuclear receptor superfamily and regulates the expression of anti-inflammatory genes.

The results shown in FIGS. 14A and 14B demonstrate the nuclear localization of the cargo, even in the presence of TNFα, which is known to induce inflammation. The results show the ability of Compound 1 to induce anti-inflammatory pathways and overcome inflammation.

Evaluation of the effect of Compound 1 on cognitive impairment after BCCI injury in rats Bilateral controlled cortical impact (BCCI) injury to the central prefrontal cortex (MFC) of male Sprague Dawley rats was induced by a cortical contusion device. After CCI, all cortical surface bleeding was controlled and the fascia and scalp were sutured. The sham-operated rats were anesthetized, placed on a stereotactic fixation device, and craniotomy was performed.

16 mg / mL progesterone was dissolved in 22.5% 2-hydroxypropyl-β-cyclodextrin and the first injection was given intraperitoneally (16 mg / kg) 1 hour after injury. The remaining injections (all 16 mg / kg) were subcutaneously administered 6 hours after the injury and continued for 5 days after the injury. Progesterone injections were made at a concentration of 1 mL / kg. Progesterone was used as a control.

Vehicles of 0.2, 0.4, and 0.6 mg / mL Compound 1 (0.6% w / v Pluronic® F-68 and 0.6% w / vPVPK-29 / 32 in water) Suspended in, 16 hours before injury and 2 hours after injury, oral administration was performed at a concentration of 10 mL / kg, and administration was continued for 4 days. At the same time, control rats were administered an injection of a vehicle equivalent of Compound 1. The treatment groups are summarized in Table 11.

The Morris Water Maze (MWM) test uses visual cues. It is a spatial course determination task that measures learning and memory in rodents. Subjects learn to discover hidden platforms over the course of several days. The MWM test is performed 2 weeks after the injury. Male Sprague Dawley rats were allowed to swim in the pool until they reached a platform located in the southwestern quadrant of the tank, or until 90 seconds had passed. Behavior was tracked by a video camera suspended above the pool and recorded and analyzed using video tracking software (ANY-maze).

The effects of Compound 1 and progesterone on the acquisition of MWM testing are shown in FIG. 15A. Two-way ANOVA has found a significant therapeutic effect. Compared to sham-injured rats, BCCI-injured rats showed significant spatial learning disabilities, indicated by a significant increase in the latency of hidden platform discovery during the 5-day acquisition phase (FIG. 15A). Compared to vehicle-treated BCCI-injured rats, Compound 1 (2, 4, and 6 mg / kg) showed a dose-dependent reduction in latency for hidden platform discovery, with significant effects of injury 17 and It was seen at 6 mg / kg after 18 days and at 4 mg / kg after 18 days. The data suggest that Compound 1 has a neuroprotective effect.

FIG. 15C is a photograph of the entire animal brain undergoing sham treatment (Sham), CCI + vehicle (control), or CCI + Compound 1 (6 mg / kg) showing the results of a qualitative visual examination of the entire brain before vibratome excision. Is. Examination suggested that none of the Sham animals (0 of 4) showed damage to the medial foot dorsal tissue. In stark contrast, all four CCI controls showed severe bilateral injury confined to this area of the cortex. CCI animals treated with Compound 1 showed moderate to minimal damage. In particular, even the brain showing the most severe injury in the compound 1 group was not so extreme as compared with all the brains in the CCI control group.

Expression levels of several cytokines were measured in plasma collected from rats in each treatment group. Samples were frozen and stored at -80 ° C. On the day of the experiment, the sample was thawed, diluted 4-fold and analyzed for cytokine expression on the Luminex platform. Samples were analyzed for the cytokines GRO / KC, IFNy, IL-1B, IL-6, IL-10, IL-12p70, and TNFa using a multilipix kit manufactured by Millipore. As shown in FIG. 15B, the same pattern of expression levels was observed between the samples. The biggest change was in IL-10. Compound 1 at 6 mg / kg reduced IL-10 compared to the vehicle-treated control group.

Traumatic injuries often provoke a secondary injury response. In most cases, the result is inflammation. The inflammatory response is driven by cytokines and chemokines and is partially transmitted by injured tissue-derived products (damage-related molecular patterns).

Multiple organ dysfunction syndrome (MODS), a little-understood syndrome in which organ function is progressively lost, most often causes late-onset mortality after injury and is a significant proportion of morbidity and mortality. Occupy. MODS is, to some extent, attributed to excessive or maladaptated activation of the inflammatory pathway.

Quantitative measurements of cell density were collected from an anti-NeuN immunolabeled subsection approximately 2-3 mm anterior to the bregma. A region of interest (ROI) was removed from the periphery of layer IV-VI in the adjacent cortical region to the site of injury in the CCI-treated animal or to the equivalent region (dorsal cortex) in the sham-treated animal (blind for experimental conditions). Inspection). Within the ventral cortex region in the same section, the ROI of a similar region was also evaluated. Cell identification was performed using the cell counting module of the Keyence BZ-II analyzer software. For each ROI, the cell-to-gray matter (CG) area factor was determined. The sham-treated animals showed uniform high density labeling both in the dorsal and ventral regions. As expected, CCI control animals were both dorsal (-45% compared to sham treatment) and ventral (-30% compared to sham treatment) cortical areas compared to sham-treated animals. The CG coefficient decreased (FIG. 15D). The CCI-induced reduction in CG coefficient was alleviated in the ventral cortex by treatment with Compound 1 (-3% compared to sham treatment; p = 0.09). The effect of Compound 1 in the ventral cortex was not statistically significant compared to vehicle treatment, but this effect is expected to break statistical significance in larger studies. No effect of Compound 1 was detected in the dorsal cortex region adjacent to the injury site (-32% compared to sham treatment). The difference between the effect on the dorsal region and the effect on the ventral region observed in Compound 1 may be a threshold effect related to the degree of injury that is inversely correlated with the distance from the injury site. Therefore, damage to the dorsal cortex region may have been too severe to be rescued by Compound 1 under these conditions.

Immunofluorescence was performed to assess the effect of TBI on several immune response pathways and to determine if Compound 1 mediates its neuroprotective effect by one or more of these pathways. Immune-fluorescent labeling for anti-rat IgG (indicator of blood-brain barrier (BBB) permeability) and TNFα (indicator of neuroinflammation) were used to examine semi-quantitative measurements of secondary injury response. All markers were imaged at 20x magnification in the cortical area surrounding the injury site in the adjacent subsection within 300 μm of the area used in the NeuN labeling assessment. At each label, the target ROI was contoured within the IV-VI adjacent layer at the site of injury (or the area corresponding to the dorsal cortex of the sham-treated animal), and the reference ROI was collected from the same cortical layer in the ventral cortex. .. Normalized fluorescence intensities were evaluated for each of the two ROIs. For all labels, the ventral cortex reference site was determined not to differ between groups (p> 0.5); therefore the target percentage relative to the reference value (IF) was determined.

Anti-rat IgG was expressed in neurons of injured tissue (indicated by arrows in FIG. 15E). FIG. 15E shows that anti-rat IgG is distributed within the neuropil of the injured area of cortical tissue. Anti-rat IgG is not present in sham-treated tissue. TNFα immunopositive cells (indicated by arrows in FIG. 15E) are clearly visible in the injured tissue surrounding the injured site in control animals. These elements are rarely present in compound 1 treated and sham treated animals. FIG. 15E shows that Compound 1 reduces the secondary injury response in rats exposed to brain injury.

Collagen-induced arthritis (CIA) test No. 2
A second CIA model was created to further investigate the effects of the compounds described herein on inflammatory biomarker pairs. In this model, the groups were named Group A (unsensitized), Group B (model; vehicle treatment), and Group C (5 mg / kg daily Compound 2). Rats in groups B and C were intradermally immunized with bovine type II collagen in IFA on day 0 and boosted on day 7. Compound 2 was orally administered to rats with CIA after the onset of arthritis (day 11). CIA onset was assessed by macroscopic scoring and foot swelling measurements. It is assessed daily for the first 5 days after sensitization (7th day) using the clinical scoring system described in Table 7 above, and then twice weekly (Monday and Thursday) until 28th day. evaluated. In addition, in all animal groups, CD45, 4 days after compound treatment (15th day of study), 10 days (21st day of study-peak of disease), and final day of study (28th day of study). Measurements of CRP, CCL2 / MCP-1, TNF-α, IL1-β, IL-6, IL-17 ELISA, and cathepsin K and elastase were performed. Furthermore, on the final day of the test (28th day of the test), three-dimensional microfault densitometry of the calcaneus was performed on a small number of representative animals from each group to quantify the bone erosion of the foot. ..

16A and 16B show that rats treated with 5 mg / kg of Compound 2 had significantly reduced joint swelling (FIG. 16B) and clinical score (FIG. 16A) compared to vehicle-treated rats. ..

17A and 17B show that rats treated with 5 mg / kg of Compound 2 had significantly reduced hindfoot bone erosion compared to vehicle-treated rats. The joint condition of animals treated with Compound 2 was comparable to that of insensitive objects. In contrast, vehicle-treated animals showed a statistically significant increase in bone erosion of the hind paw.

The Luminex® assay and ELISA were used to measure the effect of Compound 2 on the levels of pro-inflammatory cytokines and pro-inflammatory markers. Twenty-one days after the first immunization, from two rats in the model group and two rats in the compound 2 treatment group, two rats in the unsensitized group and three rats in the model group at the end of the study, Lubricant was collected from 3 rats in the compound 2 treatment group.

17C-17F show that Compound 2 showed an inhibitory effect on the production of pro-inflammatory cytokines and pro-inflammatory markers in synovial fluid samples as compared to the model group. Decreased cytokines include IL-1β, IL-6, and MCP-1, and the inflammatory marker is C-reactive protein (CRP).

The Luminex® assay was also used to measure pro-inflammatory cytokine levels in serum. Serum samples (1 mL of blood per rat) were collected 4 days after compound treatment (15th day), 10 days after compound treatment (usually disease peak-21 days), and at the end of the study (27th day). ..

FIG. 17G shows that Compound 2 showed an inhibitory effect on IL-1β production in serum samples as compared to the model group.

Experimental Autoimmune Encephalomyelitis (EAE) Model The EAE model is a recognized model for the study of human CNS demyelinating diseases such as multiple sclerosis. The effect of Compound 1 on MOG induction in the EAE mouse model of female C57B1 / 6J mice was investigated. Animals were divided into three groups, named Group I (vehicle control), Group II (dexamethasone positive control), and Group III (7.5 mg / kg compound 1). Saline, dexamethasone, and compound 1 were administered according to the schedule shown in FIG. 18A. Starting from day 0, saline and dexamethasone were administered intraperitoneally daily. Starting on the 11th day (disease onset), 7.5 mg / kg of Compound 1 was orally administered on Monday, Wednesday, and Friday for 3 consecutive weeks. The disease was a single intradermal injection of MOG emulsified in complete Freund's adjuvant (CFA) on day 0 of the test, followed by pertussis toxin (PT) on day 0 of the test and again on day 2 of the test (48 hours later). ) Induced by intraperitoneal additional immunostimulation.

As shown in FIG. 18B, the first signs of the disease were noticed on days 7-9 following MOG immunization, and the onset of the disease peak was on day 17 of the study. Dexamethasone treatment (group II) starting at day 0 at a dose of 1 mg / kg IP significantly reduced clinical scores on days 8-37 of the study compared to vehicle controls (group I). Treatment with Compound 1 (Group III) starting on day 11 at a dose of 7.5 mg / kg significantly reduced the disease score and severity. These results are shown in FIG. 18B.

Given the findings obtained under these test conditions, treatment with compound 1 at an oral dose of 7.5 mg / kg, starting on day 11 of the test, significantly reduced disease score and severity.

Example 18. Wound healing model material Mouse-C57BL / 6J mouse, male, 6-8 weeks old, SPF was obtained from Harlan Laboratories LTD. Mice were housed in sterile individual ventilation cages (IVCs) and fed free food and water. A 12h / 12h light-dark cycle, temperature control at 19-21 ° C., humidity control at 40-60%, positive pressure in the animal room, and health report management every 3 months on selected sentinels were performed.
Pig-Pig (sus crofa domestica), domestic pig (mainly Landrace X large white), female, about 60 kg, 4-5 months old, Lahav Institute of Animal Research, Kibbutz Lahav, Israel. Pigs were bred in a clean, non-SPF environment with direct free access to public tap water, and food followed standard growth chart recommendations under veterinary supervision.
Isoflurane for inhalation 99.9%, lot 6027962, Abbot Laboratories Ltd, England
Water-Water for Injection, Batch 11481012, B.I. Braun Melsungen AG, Germany
Saline-0.9% sodium chloride for injection, batch 12224012, B.I. Braun Melsungen AG, Germany
DMSO-dimethyl sulfoxide, D2650, Sigma-Aldrich Inc. , U.S. S.
Pluronic® F-68
PVP K-29 / 32

Evaluation of Effect of Systemic Administration and Topical Application of Compound 1 on C57BL Mouse Skin Wounds The effect of Compound 1 on skin wound healing was tested in a mouse longitudinal full-thickness skin incision wound model. Upon arrival, the animals were identified by ear tags, weighed and adapted to the environment for several days prior to the start of the experiment. Mice were weighed on the day of wound formation and divided into 6 experimental groups of 6 mice according to weight difference stratification randomization. Prior to surgery, mice were anesthetized with isoflurane and the animals' backs were shaved. A 20 mm full-thickness longitudinal incision was made on the animal's back (parallel to the spine) using a standard scalpel blade. Three hours after wound formation, the incision became oval due to skin elasticity and animal activity. At this stage, the widest area of the wound was measured to establish a baseline wound width. Wound healing assessment was performed by measuring the widest area of the wound. The treatment group consisted of a gavage or topical group. During the experiment, the wounds were photographed and morphological analysis was performed. At the end of the experiment 8 days after wound formation, mice were slaughtered, wound width was evaluated, and a biopsy of the wound area was taken for analysis.

The dosing solution was prepared fresh on each dosing day. Compound 1 for gavage is provided as a lyophilized powder and reconstituted in aqueous 0.6% w / v Pluronic® F-68 and 0.6% w / v PVPK-29 / 32 solutions. To make a 0.75 mg / mL storage suspension and prepare 7.5 mg / kg and 4 mg / kg working solutions, it is subsequently followed by an aqueous 0.6% w / v pluronic® F-. Diluted with 68 and 0.6% w / v PVPK-29 / 32. Compound 1 for topical application was provided as a lyophilized powder, suspended in water at a concentration of 10 mM and further diluted with water to the final working concentration for topical application.

Photograph recording was performed using the digital camera FinePix S700 as part of the daily morphological evaluation. FIG. 19 is a photograph of a typical wound from each experimental group 5 days after wound formation. The black bar scale represents 1 cm. A total of 33 wounds were made in 33 mice. Morphological evaluation demonstrated the favorable effects of treatment with Compound 1, both oral and topical. All treatments induced better wound healing than controls. The treated wounds were smaller in size and the scabs were lighter, thinner and more homogeneous, without cracks, suggesting late wound healing. Control group wounds evaluated on the same day as the treatment group appear to be larger in size, exposing unhealed wound areas (observed as reddish pink areas) both at the edges and in the center of the wound. It was covered with thick, cracked scabs.

Morphological analysis is a key parameter used in wound healing assessments in preclinical animal studies and in clinical treatment of human wounds. Based on morphological analysis, Compound 1 showed efficacy and had a positive effect on wound healing. It should be noted that both topical and systemic administration of Compound 1 resulted in better wound healing, as determined by reduced wound size and better crusting properties.

Assessment of the Effect of Topical Application of the Test Compound on Pig Skin Wounds The effect of the test compound on skin wound healing can be tested in a pig longitudinal full-thickness skin incision wound model. Upon arrival, animals are identified by ear tags, weighed and adapted to the environment for several days prior to the start of the experiment. Three days before surgery, the pigs are transferred to an inpatient facility for acclimatization. Stop feeding 12 hours before treatment. On the day of surgery, pigs are anesthetized with ketamine, xylazine, diazepam, and isoflurane. Carefully trim the dorsal thorax and abdominal coat using an Oster® shaving machine (blade size 30) and label each of the 20 individual areas of 4 cm ² in two rows. (10 areas per row). Using the # 11 scalpel blade, make 10 pairs of 2.5 cm full-thickness longitudinal skin incisions 4 cm from both sides of the dorsal midline.

Following surgery, the wound is divided into experimental groups and treated daily by topical application to the wound area and wound margin. The treatment area consists of the surface of the skin up to a distance of 2 cm from the center of the wound. The dosage solution is applied little by little to each wound using a pipette until the entire therapeutic volume (eg 1 mL saline or test compound) is absorbed by the tissue.

Hours after wound formation, the incision becomes oval due to the elasticity of the skin and the activity of the animal. At this stage, the widest area of the wound is measured to establish a baseline wound width. Wound healing assessment is performed by measuring the widest area of the wound. During the experiment, the wound is photographed and a morphological analysis is performed.

At the end of the experiment (eg 12 days after wound formation), anesthetics and KCl are administered to kill the pigs. Wound morphology is evaluated, wound width is measured, a biopsy of the wound area is taken and fixed using 4% paraformaldehyde for further analysis. Following immobilization, the wound biopsy is photographed using a high resolution digital camera such as the FinePix S700 and the biopsy of the wound area is subjected to histopathological analysis. Assessment of wound healing is performed in a paired fashion in which each wound treated with the test compound is directly compared to a control wound at the same anatomical location opposite the dorsal midline. Due to the variability associated with the degree of skin angiogenesis and blood circulation in different areas of the pig's back, this combined assessment of healing is from the perspective of objective assessment and objective comparison of treated and untreated wounds. is important. Frontal wounds near the neck exhibit much better healing properties than wounds located posteriorly.

The dosing solution should be prepared fresh on each dosing day. The test compound is provided as a lyophilized powder and further reconstituted with 0.9% sodium chloride for injection to make a 3 mg / mL storage suspension. The storage suspension is further diluted with 0.9% sodium chloride for injection to final concentrations of 3 μM and 1 μM for topical application.

A homogeneous, thin, uniformly organized scab surface with no leakage, bleeding or secretory events from the wound suggests wound healing. Very heterogeneous, cracked, dark crusts suggest numerous exudative, leaky, and bleeding events during the wound healing process.

Evaluation of the Effect of Topical Application of the Test Compound on the Initial Wound Healing Process in Pigs The effect of the test compound on initial wound healing can be tested in a wound model with a longitudinal full-thickness skin incision. On the anterior portion of the anesthetized pig's back, a # 11 scalpel blade is used to make 5 pairs of 2.5 cm longitudinal full-thickness incisions 4 cm from both sides of the dorsal midline. Within hours after the procedure, the longitudinal incision becomes an oval wound.

Wounds are divided into experimental groups and treated daily by topical application to the wound area (including the rim and the skin area near the wound). The treatment phase begins 24 hours after wound formation. The dosing solution is applied little by little to each wound using a pipette until the entire treatment volume (eg 1 mL saline or test compound) is absorbed by the tissue.

On day 5, the condition and morphology of wound healing is assessed according to the following parameters: bleeding, leakage, swelling, inflammation, purulent secretion, and crusting. The evaluation is performed in a mating fashion in which each wound treated with the test compound is directly compared to a control wound at the same anatomical location opposite the dorsal midline.

The dosing solution should be prepared fresh on each dosing day. The test compound is provided as a lyophilized powder and reconstituted with 0.9% sodium chloride into a 3 mg / mL storage suspension. This storage suspension is further diluted with 0.9% sodium chloride to prepare the final 3 μM topical solution.

Morphological wound healing assessment is performed on the 5th day of treatment. The swelling is examined, scored according to the severity of each wound and recorded as mild, moderate or severe. Wounds showing moderate and severe swelling are presented as a percentage of all wounds in the experimental group. Secretions are tested and scored in binary mode: Wounds with minimal secretion are considered positive for this parameter and wounds without any detectable secretion are considered negative. Wounds showing secretions (positive for this parameter) are presented as a percentage of all wounds in the experimental group. The scab is a continuous layer of hard, dry, reddish dark yellow or brown structure that covers the entire wound area and adheres strongly to the wound bed, thus being continuous between the external environment and the injured tissue. It is considered to be fully formed if it provides a strong barrier. Crusting is tested and scored in binary mode: Wounds showing dry, strong, fully formed scabs are considered positive for this parameter and have no scabs or early scabs. Some wounds are considered negative. Wounds with fully formed scabs are presented as a percentage of total wounds per experimental group.

Swelling, secretion, and crusting are also assessed. Swelling and secretion are part of an excessive inflammatory response that may delay tissue repair and induce non-aesthetic scarring.

Evaluation of the effect of topical application of the test compound on early wound healing of porcine skin and on hypersensitivity and scratching behavior associated with damaged or injured skin The effect of the test compound on skin wound healing is a longitudinal full-thickness skin incision in pigs. Can be tested with a wound model. Three days before surgery, the pigs are transferred to an inpatient facility for acclimatization. Stop feeding 12 hours before surgery. On the day of surgery, pigs are anesthetized with ketamine, xylazine, diazepam, and isoflurane. The Oster® hair shaving machine (blade size 30) was used to cut the dorsal thorax and abdominal hair. Mark 10 pairs of 4 cm ² sections each and use a # 11 scalpel blade to make a 2.5 cm full-thickness longitudinal skin incision on either side of the dorsal midline.

Following surgery, the wound is divided into experimental groups and treated daily by topical application to the wound area, including the edges and the nearby skin area up to a distance of 2 cm in all directions from the wound. The dosing solution is applied little by little to each wound using a pipette until the entire treatment volume (eg 1 mL vehicle or test compound) is absorbed by the tissue.

Within hours after the procedure, the longitudinal incision becomes an oval wound due to cutaneous elasticity and animal activity. During the experiment, the wound is photographed and a morphological analysis is performed. Assessment of wound healing is performed in a paired fashion in which each wound treated with the test compound is directly compared to a control wound at the same anatomical location opposite the dorsal midline. Wound morphology and animal behavior are recorded for the first 5 days following wound formation.

The dosing solution should be prepared fresh on each dosing day. The test compound is provided as a lyophilized powder and is dissolved in 100% DMSO at a stock solution concentration of 15 mM. Further dilution with water for injection is performed to final concentrations of 3 μM and 1 μM for topical application.

As part of the daily morphological evaluation, wound photography is performed using, for example, the digital high resolution camera FinePix S700. In addition to the wound condition, observe the inflamed and scratched skin area. Scratching behavior usually does not cause damage to the wound, or wound healing, because the wound is created on the dorsal surface near the dorsal midline, as animals are difficult and nearly impossible to reach. Does not interfere with the process.

Evaluation of the effects of Compound 1 in PVP / Pluronic® F-68 and Compound 1 in water on scratching behavior associated with skin healing in mice In the skin wound tests in mice described herein, animal behavior. We also observed and quantified scar removal attempts, signs of discomfort, and scratching behavior in the wound area. Abnormal behavior, presentation of abnormal scratching behavior, and signs of pain from all studies conducted in mice were analyzed. In these tests, treatment was performed using Compound 1 in PVP / Pluronic® F-68, Compound 1 in water, and each vehicle control.

During all wound healing experiments in mice, monitoring of healing parameters, signs of skin hypersensitivity, and other skin conditions associated with wound healing is performed in the area near the wound and in the treated skin area. In addition, special attention was paid to animal behavior such as signs of discomfort and pain; and signs of scratching and over-tampering of wounds and skin during the treatment phase of all skin healing models. The palliative and soothing effects of the treatment compounds were very pronounced compared to control animals, which tended to tamper with their wounds.

In mice, treatment with Compound 1 in PVP / Pluronic® F-68 or Compound 1 in water was wound compared to vehicle-treated mice (DMSO in water, saline, PVP / Pluronic or water). Reduced over-tampering.

In mice, over-tampering with the wound resulted in eschar removal and bleeding, or damage to newly formed tissue in the wound bed that adhered strongly to the scab. Most of these events occurred in the vehicle-treated group (about 20-30% of all experiments).

According to a summary of skin conditions and animal behavior, wound treatment with Compound 1 in PVP / Pluronic® F-68 or Compound 1 in water is likely to be some of the treatment compounds for damaged and inflamed skin. It can be concluded that it prevents over-tampering of wounds in mice for its palliative and soothing effects.

Test Compound Dose Response for Skin Wound Healing The effect of the test compound on skin wound healing can be tested in a mouse longitudinal full-thickness skin incision wound model. Upon arrival, animals are identified by ear tags, weighed and adapted to the environment for several days prior to the start of the experiment. Mice are weighed on the day of wound formation and divided into 7 experimental groups (N = 6 or N = 7) according to weight difference stratification randomization. The vehicle group was administered 0.1% DMSO in water, while the positive control group was aqueous 0.6% w / v Pluronic® F-68 and 0.6% w / v PVPK-29 / 32. Treat with. Prior to surgery, mice are anesthetized with isoflurane and the animal's back is trimmed. A 20 mm full-thickness longitudinal incision is made on the animal's back (parallel to the spine) using a standard scalpel blade. Three hours after wound formation, the incision becomes oval due to the elasticity of the skin and the activity of the animal. At this stage, the widest area of the wound is measured to establish a baseline wound width. Wound healing assessment is performed by measuring the widest area of the wound. Wound treatment is performed by topical application of the administration solution (eg 0.2 mL) directly (daily) to the wound.

The dosing solution should be prepared fresh on each dosing day. The test compound is provided as a lyophilized powder and returned to a 3 mg / mL storage suspension with 0.1% DMSO in water. The storage suspension is further diluted with 0.1% DMSO in water to prepare the final topical solution. Control wounds were treated topically with 0.1% DMSO in water or 0.6% w / v Pluronic® F-68 and 0.6% w / v PVPK-29 / 32 solutions in water. Will be done.

At the end of the experiment 8 days after wound formation, mice are killed by CO ₂ inhalation, wound width is measured, and a biopsy of the wound area is taken for histological analysis. Fix the biopsy using 4% paraformaldehyde. Following immobilization of the entire wound area, a 5 mm incision is made in the widest area of the wound and these specimens are paraffin-embedded. Paraffin blocks are prepared using standard procedures for stepwise dehydration of tissue and paraffin embedding. Histological sections are then prepared and the tissue stained with hematoxylin and eosin (H & E) staining. H & E staining slides are examined to perform an assessment of wound healing efficacy.

Advanced skin closure is assessed on day 8 by examination of healthy eosin-stained dermis and newly formed dermal margins in the wound space. Wounds in which both dermal margins are observed in a 100x field of view under a microscope (BX41 Olympus or Axiovert 25, Zeiss) are considered positive for advanced skin closure healing parameters. The number of wounds with advanced skin closure is presented as a percentage of all wounds in the experimental group.

Advanced epidermal closure is assessed on day 8 using H & E staining by analyzing tissue sections of the widest area of the wound. Wounds showing the presence of a continuous layer of epidermis covering the entire wound gap and wounds with the most advanced epidermal marginal movement, observed at 400x field of view under a microscope, are considered positive for advanced skin closure parameters. Results are presented as a total percentage per experimental group.

Epidermal migration is assessed on day 8 by analyzing the newly formed epidermis stained with concentrated hematoxylin on both wound edges using H & E staining. The epidermal margin is considered mobile if the newly formed epidermal margin covers 20-30% of the wound gap. The mobile epidermal margins in the group are counted and presented as a percentage of the total number of epidermal margins (twice the number of wounds in the group). Both epidermal margins are considered migratory in wounds that exhibit complete or advanced epidermal closure. A total of 62 wounds are made in 62 mice.

Wound treatment with the test compound prevents wound healing complications such as epidermal hyperplasia and adhesions The effect of the test compound on skin wound healing can be tested in a mouse longitudinal full-thickness skin incision wound model. Prior to surgery, mice are anesthetized with isoflurane and the animal's back is shaved. A 20 mm full-thickness longitudinal incision is made on the animal's back (parallel to the spine) using the blade of a scalpel. Three hours after wound formation, the incision becomes oval due to the elasticity of the skin and the activity of the animal. Wound healing assessment is performed by measuring the widest area of the wound. For example, 0.2 mL of test compound is locally applied directly to the wound daily to perform wound treatment. The wound care process is a partially moist environment and after daily treatment the wound is moist for some time. At the end of the experiment 8 days after wound formation, the mice are slaughtered, the wound width is evaluated, and a biopsy of the wound area is taken for analysis.

The dosing solution should be prepared fresh on each dosing day. The test compound is provided as a lyophilized powder and returned to a 3 mg / mL storage suspension with 0.1% DMSO in water and subsequently diluted with 0.1% DMSO in water to the concentration used for topical application. To do. Wounds in the control group were treated topically with 0.1% DMSO in water, or 0.6% w / v Pluronic® F-68 and 0.6% w / v PVPK-29 / 32 in water. To.

At the end of the treatment phase 8 days after wound formation, mice are killed by CO ₂ inhalation and a biopsy of the wound area is taken. Immobilization of wound tissue is performed using 4% paraformaldehyde. Following immobilization of the entire wound area, a 5 mm incision is made in the widest area of the wound and paraffin-embedded. Paraffin blocks are prepared using standard procedures for stepwise dehydration. Histological sections are then prepared and the tissue stained with hematoxylin and eosin (H & E). Evaluate and graph wound healing parameters.

Epidermal hyperplasia is assessed on day 8. Non-migratory and hyperplastic epidermal margins in the group are counted and presented as a percentage of the total number of epidermal margins (twice the number of wounds in the group). Hyperplastic epidermal margins are assessed using H & E staining by analyzing concentrated hematoxylin-stained areas of the epidermis. If the epidermal margin appears to be thicker than the normal epidermis of a healthy skin area and such epidermal margin does not show migration towards wound gap closure, it is considered hyperplastic and immobile. ..

Wound gap adhesions are assessed on day 8. Adhesions are assessed by analysis of cell and tissue structure in the wound space. Wound adhesions are scored on a mild, moderate or severe scale. A negative score is considered if there is a clot in the wound space or if normal granulation tissue is replaced by other tissue such as skeletal muscle or extensive lymphoid tissue. Some adhesions or abnormal granulation that occupy more than 40% of the wound gap area are considered severe. Adhesion is considered mild if it is not prominent and does not interfere with normal skin tissue regeneration. Wounds with severe adhesions are calculated as a percentage of total wounds per experimental group and graphed as shown. A total of 32 wounds (64 epidermal margins) are produced in 32 mice.

One of the most important wound healing complications is epidermal hyperplasia. In response to the stress signals associated with wound formation, cell proliferation in the basal layer of the epidermis compensates for skin loss. Under normal conditions, in peaceful and safe wound healing, epidermal cells begin to migrate towards the closure of the wound gap shortly after proliferation. If migration does not occur or is delayed, such as skin complications caused by hyperglycemia in diabetic wounds, epidermal hyperplasia becomes prominent and may cause further complications in wound healing. In acute open wounds such as the model used in this experiment, or in acute suture wounds such as postoperative wounds, reduced epidermal healing associated with epidermal marginal hyperplasia is associated with a risk of contamination, and wound dehiscence, from the wound. Increases the risk of fluid drainage or other wound healing complications such as tissue protrusion from the wound.

In an effective wound healing process, the primary blood clot undergoes a gradual change to produce granulation tissue in the wound space, which, following remodeling, is finally formed into a fully restored function. Becomes skin tissue. Adhesion of non-skin-related tissue in the wound space results in poor formation of granulation tissue, resulting in limited final tissue remodeling. This may cause further restrictions on the functioning of healing skin.

Wound treatment with a test compound in a saline-based formulation improves wound healing and prevents severe adhesions The effect of the test compound on skin wound healing has been tested in a mouse longitudinal full-thickness skin incision wound model. It was. Surgery is performed on 7-8 week old C57BL male mice anesthetized with isoflurane. Prior to surgery, mice are anesthetized with isoflurane and shaved. A 20 mm full-thickness longitudinal incision is made using a standard scalpel blade. Three hours after wound formation, the incision becomes oval due to the elasticity of the skin and the activity of the animal. At this stage, the widest area of the wound is measured to establish a baseline wound width. Wound healing assessment is performed by measuring the widest area of the wound. Wound treatment is performed by topically applying 0.2 mL of solution directly to the wound daily. After daily treatment, the wound is moist for some time (3-5 hours), so the wound care process is performed in a partially moist environment. At the end of the experiment 8 days after wound formation, the mice are slaughtered, the wound width is evaluated, and a biopsy of the wound area is taken for analysis.

The dosing solution should be prepared fresh on each dosing day. The test compound is provided as a lyophilized powder and reconstituted with saline to make a 3 mg / mL storage suspension, which is subsequently diluted with saline to a working concentration of 3 μM for topical application. Control wounds are treated topically with saline.

At the end of the treatment phase 8 days after wound formation, mice are killed by CO ₂ inhalation and a biopsy of the wound area is taken. Immobilization of wound tissue is performed using 4% paraformaldehyde. Following immobilization of the entire wound area, a 5 mm incision is made in the widest area of the wound and the incision area is paraffin-embedded. Paraffin blocks are prepared using standard procedures for stepwise dehydration. Histological sections are then prepared and the tissue stained with hematoxylin and eosin (H & E). Evaluate and graph wound healing parameters.

Epidermal closure is assessed by analyzing tissue sections in the widest area of the wound using H & E staining. Wounds showing the presence of a continuous layer of epidermis covering the entire wound gap and wounds with the most advanced epidermal edge movement, observed at 400x field of view under a microscope, are considered positive for advanced skin closure parameters. Results are presented as a total percentage per experimental group.

Skin healing is assessed by examination of healthy eosin-stained dermis and newly formed dermal margins in the wound space. Wounds in which both dermal margins are observed in a 100x field of view under a microscope (BX41 Olympus or Axiovert 25, Zeiss) are considered positive for advanced skin closure healing parameters. The number of wounds with advanced skin closure is presented as a percentage of all wounds in the experimental group.

Granulation tissue is evaluated using H & E staining. Granulation tissue is considered early when the primary fibrin thrombus is replaced by fibrous connective tissue containing adipocytes, new capillaries, and infiltrates containing lymphoid cells, macrophages and plasma cells. Early granulation tissue replaced with tissue with a large number of fibroblasts and collagen fibers is considered late. Overall, the late granulation tissue region of the wound gap is recorded as a percentage of the total wound gap region. Wound gaps that present late granulation tissue formation covering 40% of the wound gaps are considered positive for this parameter. Results are calculated as a percentage of total wounds per group.

Adhesions are assessed by analysis of cell and tissue structure in the wound space. Wound adhesions are scored on a mild, moderate or severe scale. A negative score is considered if there is a clot in the wound space or if normal granulation tissue is replaced by other tissue such as skeletal muscle or extensive lymphoid tissue. Some adhesions or abnormal granulation that occupy more than 40% of the wound gap area are considered severe. Adhesion is considered mild if it is not prominent and does not interfere with normal skin tissue regeneration. Wounds with severe adhesions are calculated as a percentage of total wounds per experimental group. In 19 mice, 19 wounds (38 epidermal margins) are created.

Evaluation of the effect of topical application of the test compound on the late wound healing process and scars on porcine skin The effect of the test compound on late skin wound healing will be investigated in a porcine wound model with a longitudinal full-thickness incision. On the day of surgery, pigs are anesthetized with ketamine, xylazine, diazepam, and isoflurane. Cut the dorsal thorax and abdominal coat and use a # 11 scalpel blade, 4 cm from both sides of the dorsal midline, 10 pairs of 2.5 cm full-thickness longitudinal Make a skin incision. Following surgery, the wound is divided into experimental groups and treated daily by topical application to the wound area and wound margin, including treatment of the skin near the wound area up to a distance of 2 cm in all directions from the wound center. To do. Dosage is applied little by little to each wound using a pipette until the entire therapeutic volume (eg 1 mL vehicle or test compound) is absorbed by the tissue. Skin near the wound is treated with gauze soaked in test compound or vehicle solution.

During the experiment, the wound is photographed and a morphological analysis is performed. At the end of the treatment phase (19 days after wound formation), pigs are slaughtered by administration of anesthetics and KCl. The morphology of the wound is examined, the wound is photographed, and a biopsy of the wound area is taken for immobilization and further morphological and histological analysis.

The dosing solution should be prepared fresh on each dosing day. The test compound is provided as a lyophilized powder and dissolved in 100% DMSO to prepare a stock solution concentration of 15 mM. Subsequently, further dilution with water for injection is performed to a final concentration of 3 μM and 1 μM for topical application.

At the end of the treatment phase (19 days after wound formation), a wound healing assessment will be performed. Completely healed wounds are reported as a percentage of total wounds per group. The average width of scars in wounds that heal completely and show complete crusting is also reported. Scars were measured (mm) and the mean width and standard deviation of the scars were calculated. A total of 20 wounds were performed.

At the end of the treatment phase (19 days after wound formation), pigs are slaughtered by overdose of anesthetic and KCl and a biopsy of the wound area is taken. Wound biopsy immobilization is performed using 4% paraformaldehyde. Following immobilization, the wound biopsy is photographed at maximum resolution using, for example, the digital camera FinePix S700.

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The teachings of all patents, publications, and references cited herein are incorporated by reference in their entirety.

While the present invention has been specifically demonstrated and described by reference to examples of embodiments thereof, various modifications in the form and details are made without departing from the scope of the present invention within the scope of the appended claims. Those skilled in the art will appreciate that it may be added.

Claims (15)

Formula I:

Compound (in the formula,
R ¹ is selected from hydrogen and C _{1 to} C ₄ alkyl;
R ² is selected from O and S;
R ³ is -N (R ⁴ )-(C _{3 to} C ₆ cycloalkyl), -C _{3 to} C ₆ alkyl,-(C _{0 to} C ₁ alkylene) -heterocyclyl, and-(C _{0 to} C ₄ alkylene). ) - is selected from heteroaryl, any alkyl or alkylene down unit content of R ³ are, -N ^(R _{5) 2} (wherein each ^{R 5} is independently selected from hydrogen and _C 1 -C ₄ alkyl optionally substituted with);
Any heterocyclyl and heteroaryl portion of R ³ is, comprises at least one nitrogen atom in the ring;
Any heterocyclyl and heteroaryl portion of R ³ _is optionally substituted with C 1 -C ₄ alkyl;
R ⁴ is, Ru hydrogen der)
Or its pharmaceutically acceptable salt, solvate or hydrate, and
Pharmaceutically acceptable carrier
An orally acceptable dosage form formulation comprising . R ¹ is selected from hydrogen and methyl, orally acceptable dosage form of a formulation according to claim 1. The orally acceptable dosage form of the formulation according to claim 2, wherein R ¹ is hydrogen. The preparation of an orally acceptable dosage form according to any one of claims 1 to 3, wherein R ² is O. The preparation of an orally acceptable dosage form according to claim 1 , wherein R ³ is-(C _{0 to} C ₁ alkylene) -heterocyclyl. The preparation of an orally acceptable dosage form according to claim 5 , wherein R ³ is-(C ₁ alkylene) -heterocyclyl. The preparation of an orally acceptable dosage form according to claim 5 or 6 , wherein the heterocyclyl is selected from pyrazineyl, piperidinyl, morpholinyl, and pyrazolyl. Wherein heterocyclyl is a model Ruhorini Le, orally acceptable dosage form of a formulation according to claim 7. R ³ is -C (CH ₃ ) ₃ , -CH (NH ₂ ) -CH (CH ₃ ) ₂ , -NH-cyclopropyl,-(CH ₂ ) _0-1 -pyrazinyl, piperidinyl, hydroxypiperidinyl, N- methylpiperidinyl, -CH 2 _- morpholin-4-yl, and are selected from methyl pyrazolyl, orally acceptable dosage form of a formulation according to claim 1. R ³ is -C (CH ₃ ) ₃ , -CH (NH ₂ ) -CH (CH ₃ ) ₂ , -NH-cyclopropyl,-(CH ₂ ) _0-1 -pyrazine-2-yl, piperidine-3 The preparation of the orally acceptable dosage form according to claim 9 , which is selected from -yl, -CH ₂ -morpholin-4-yl, and 5-methyl-1-H-pyrazole-4-yl. R ³ is -C (CH ₃ ) ₃ , -NH-cyclopropyl, -CH ₂ -pyrazine-2-yl, -pyrazine-2-yl, -CH ₂ -morpholine-4-yl, and 5-methyl- The orally acceptable dosage form formulation of claim 10 , selected from 1-H-pyrazole-4-yl. Structural formula shown below :

Either the compound monomers other represented by one orally acceptable dosage form of a formulation comprising a pharmaceutically acceptable salt thereof. The preparation of an orally acceptable dosage form according to claim 12 , wherein the compound is selected from any one of compounds 1, 2, 3, 4, 8, 11, 12, and 13. Increasing fertilizing disease, angiogenic diseases, inflammatory diseases, autoimmune disorders, viral infections, wounds, ophthalmic diseases, neurodegenerative diseases, abnormal tissue growth disorders, food intake related disorders, allergies, stroke, traumatic brain injury and for the treatment of a disease selected from respiratory diseases, orally acceptable dosage form of a formulation according to claim 1. The orally acceptable dosage form formulation according to claim 1 for the treatment of neurodegenerative diseases.